84P2A9: a prostate and testis specific protein highly expressed in prostate cancer

ABSTRACT

A novel gene (designated 84P2A9) and its encoded protein is described. While 84P2A9 exhibits prostate and testis specific expression in normal adult tissue, it is aberrantly expressed multiple cancers including prostate, testis, kidney, brain, bone, skin, ovarian, breast, pancreas, colon, lymphocytic and lung cancers. Consequently, 84P2A9 provides a diagnostic and/or therapeutic target for cancers, and the 84P2A9 gene or fragment thereof, or its encoded protein or a fragment thereof used to elicit an immune response.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of co-pending U.S. patent applicationSer. No. 09/771,312, which claims the benefit of U.S. provisional patentapplication No. 60/178,560, filed Jan. 26, 2000, the entire contents ofwhich are incorporated herein by reference.

FIELD OF THE INVENTION

The invention described herein relates to a novel gene and its encodedprotein, termed 84P2A9, and to diagnostic and therapeutic methods andcompositions useful in the management of various cancers that express84P2A9, particularly prostate cancers.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of human death next to coronarydisease. Worldwide, millions of people die from cancer every year. Inthe United States alone, cancer causes the death of well over ahalf-million people annually, with some 1.4 million new cases diagnosedper year. While deaths from heart disease have been decliningsignificantly, those resulting from cancer generally are on the rise. Inthe early part of the next century, cancer is predicted to become theleading cause of death.

Worldwide, several cancers stand out as the leading killers. Inparticular, carcinomas of the lung, prostate, breast, colon, pancreas,and ovary represent the primary causes of cancer death. These andvirtually all other carcinomas share a common lethal feature. With veryfew exceptions, metastatic disease from a carcinoma is fatal. Moreover,even for those cancer patients who initially survive their primarycancers, common experience has shown that their lives are dramaticallyaltered. Many cancer patients experience strong anxieties driven by theawareness of the potential for recurrence or treatment failure. Manycancer patients experience physical debilitations following treatment.Furthermore, many cancer patients experience a recurrence.

Worldwide, prostate cancer is the fourth most prevalent cancer in men.In North America and Northern Europe, it is by far the most commoncancer in males and is the second leading cause of cancer death in men.In the United States alone, well over 40,000 men die annually of thisdisease—second only to lung cancer. Despite the magnitude of thesefigures, there is still no effective treatment for metastatic prostatecancer. Surgical prostatectomy, radiation therapy, hormone ablationtherapy, surgical castration and chemotherapy continue to be the maintreatment modalities. Unfortunately, these treatments are ineffectivefor many and are often associated with undesirable consequences.

On the diagnostic front, the lack of a prostate tumor marker that canaccurately detect early-stage, localized tumors remains a significantlimitation in the diagnosis and management of this disease. Although theserum prostate specific antigen (PSA) assay has been a very useful toolhowever its specificity and general utility is widely regarded aslacking in several important respects.

Progress in identifying additional specific markers for prostate cancerhas been improved by the generation of prostate cancer xenografts thatcan recapitulate different stages of the disease in mice. The LAPC assAngeles Prostate Cancer) xenografts are prostate cancer xenografts thathave survived passage in severe combined immune deficient (SCID) miceand have exhibited the capacity to mimic the transition from androgendependence to androgen independence (Klein et al., 1997, Nat. Med.3:402). More recently identified prostate cancer markers include PCTA-1(Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252),prostate-specific membrane (PSM) antigen (Pinto et al., Clin Cancer Res1996 September; 2(9):1445-51), STEAP (Proc Natl Acad Sci USA. 1999 Dec.7; 96(25):14523-8) and prostate stem cell antigen (PSCA) (Reiter et al.,1998, Proc. Natl. Acad. Sci. USA 95: 1735).

While previously identified markers such as PSA, PSM, PCTA and PSCA havefacilitated efforts to diagnose and treat prostate cancer, there is needfor the identification of additional markers and therapeutic targets forprostate and related cancers in order to further improve diagnosis andtherapy.

SUMMARY OF THE INVENTION

The present invention relates to a novel, largely prostate andtestis-related gene, designated 84P2A9, that is over-expressed inmultiple cancers including prostate, testis, kidney, brain, bone, skin,ovarian, breast, pancreas, colon, lymphocytic and lung cancers. Northernblot expression analysis of 84P2A9 gene expression in normal tissuesshows a highly prostate and testis-related expression pattern in adulttissues. Analysis of 84P2A9 expression in normal prostate and prostatetumor xenografts shows over-expression in LAPC-4 and LAPC-9 prostatetumor xenografts, with the highest expression in LAPC-9. The nucleotide(SEQ ID NO: 1) and amino acid (SEQ ID NO: 2) sequences of 84P2A9 areshown in FIG. 2. Portions of the 84P2A9 amino acid sequence show somehomologies to ESTs in the dbEST database. The prostate andtestis-related expression profile of 84P2A9 in normal adult tissues,combined with the over-expression observed in prostate tumor xenografts,shows that 84P2A9 is aberrantly over-expressed in at least some cancers,and thus serves as a useful diagnostic and/or therapeutic target forcancers such as prostate, testis, kidney, brain, bone, skin, ovarian,breast, pancreas, colon, lymphocytic and lung cancers (see, e.g., FIGS.4-8).

The invention provides polynucleotides corresponding or complementary toall or part of the 84P2A9 genes, mRNAs, and/or coding sequences,preferably in isolated form, including polynucleotides encoding 84P2A9proteins and fragments of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 ormore amino acids, DNA, RNA, DNA/RNA hybrids, and related molecules,polynucleotides or oligonucleotides complementary or having at least a90% homology to the 84P2A9 genes or mRNA sequences or parts thereof, andpolynucleotides or oligonucleotides that hybridize to the 84P2A9 genes,mRNAs, or to 84P2A9-encoding polynucleotides. Also provided are meansfor isolating cDNAs and the genes encoding 84P2A9. Recombinant DNAmolecules containing 84P2A9 polynucleotides, cells transformed ortransduced with such molecules, and host-vector systems for theexpression of 84P2A9 gene products are also provided. The inventionfurther provides antibodies that bind to 84P2A9 proteins and polypeptidefragments thereof, including polyclonal and monoclonal antibodies,murine and other mammalian antibodies, chimeric antibodies, humanizedand fully human antibodies, and antibodies labeled with a detectablemarker.

The invention further provides methods for detecting the presence andstatus of 84P2A9 polynucleotides and proteins in various biologicalsamples, as well as methods for identifying cells that express 84P2A9. Atypical embodiment of this invention provides methods for monitoring84P2A9 gene products in a tissue or hematology sample having orsuspected of having some form of growth disregulation such as cancer.

The invention further provides various immunogenic or therapeuticcompositions and strategies for treating cancers that express 84P2A9such as prostate cancers, including therapies aimed at inhibiting thetranscription, translation, processing or function of 84P2A9 as well ascancer vaccines.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the 84P2A9 suppression subtractive hybridization (SSH) DNAsequence of about 425 nucleotides in length (SEQ ID NO: 3). Thissequence was identified in comparisons of cDNAs from various androgendependent and androgen independent LAPC xenografts.

FIG. 2 (FIGS. 2-1, 2-2, 2-3) shows the nucleotide (SEQ ID NO: 1) andamino acid (SEQ ID NO: 2) sequences of 84P2A9-clone 1 ds sequence andORF. See Example 2, infra. The sequence surrounding the start ATG (AACATG G) (SEQ ID NO: 4) exhibits a Kozak sequence (A at position −3, and Gat position +1). The start methionine with Kozak sequence is indicatedin bold, the nuclear localization signals are boxed.

FIGS. 3A and 3B show the amino acid sequence alignment of 84P2A9 (SEQ IDNO: 2) with KIAA1552 (SEQ ID NO: 5) and LUCA15 (SEQ ID NO: 6). FIG. 3Ashows that the 84P2A9 protein sequence (bottom line) has some homologyto the human brain protein KIAA1152 (39.5% identity over a 337 aminoacid region, Score: 407.0; Gap frequency: 5.9%). FIG. 3B shows that the84P2A9 protein sequence (bottom line) contains a domain that ishomologous to a portion of the LUCA15 tumor suppressor protein (64.3%identity over a 42 amino acid region, Score: 138.0; Gap frequency:0.0%).

FIGS. 4A-4C show the Northern blot analysis of the restricted 84P2A9expression in various normal human tissues (using the 84P2A9 SSHfragment as a probe) and LAPC xenografts. Two multiple tissue northernblots (Clontech) (FIGS. 4A and 4B) and a xenograft northern blot (FIG.4C) were probed with the 84P2A9 SSH fragment. Lanes 1-8 in FIG. 4Aconsist of mRNA from heart, brain, placenta, lung, liver, skeletalmuscle, kidney and pancreas respectively. Lanes 1-8 in FIG. 4B consistof total RNA from spleen, thymus, prostate, testis, ovary, smallintestine, colon and leukocytes respectively. Lanes 1-5 in FIG. 4Cconsist of mRNA from prostate, LAPC-4 AD, LAPC-4 AI, LAPC-9 AD andLAPC-9 AI respectively. Size standards in kilobases (kb) are indicatedon the side. Each lane contains 2 μg of mRNA for the normal tissues and10 μg of total RNA for the xenograft tissues. The results show theexpression of 84P2A9 in testis and prostate and the LAPC xenografts.

FIGS. 5A, 5B, and 5C show the Northern blot analysis of 84P2A9expression in prostate and multiple cancer cell lines. Lanes 1-56 showexpression in LAPC-4 AD, LAPC4 AI, LAPC-9 AD, LAPC-9 AI, LNCaP, PC-3,DU145, TsuPr1, LAPC-4 CL, HT1197, SCaBER, UM-UC-3, TCCSUP, J82, 5637,293T, RD-ES, PANC-1, BxPC-3, HPAC, Capan-1, SK-CO-1, CaCo-2, LoVo, T84,Colo-205, KCL 22, PFSK-1, T98G, SK-ES-1, HOS, U2-OS, RD-ES, CALU-1,A427, NCI-H82, NCI-H146, 769-P, A498, CAKI-1, SW839, BT20, CAMA-1,DU4475, MCF-7, MDA-MB-435s, NTERRA-2, NCCIT, TERA-1, TERA-2, A431, HeLa,OV-1063, PA-1, SW626 and CAOV-3 respectively. High levels of 84P2A9expression were detected in brain (PFSK-1, T98G), bone (HOS, U2-OS),lung (CALU-1, NCI-H82, NCI-H146), and kidney (769-P, A498, CAKI-1,SW839) cancer cell lines. Moderate expression levels were detected inseveral pancreatic (PANC-1, BxPC-3, HPAC, CAPAN-1), colon (SK-CO-1,CACO-2, LOVO, COLO-205), bone (SK-ES-1, RD-ES), breast (MCF-7,MDA-MB-435s) and testicular cancer (NCCIT) cell lines.

FIG. 6 shows the Northern blot analysis of 84P2A9 expression in prostatecancer patient samples. Prostate cancer patient samples show expressionof 84P2A9 in both the normal and the tumor part of the prostate tissues.Lanes 1-7 show Normal prostate, Normal prostate, Patient 1 normaladjacent tissue, Patient 1 Gleason 9 tumor, Patient 2 normal adjacenttissue, Patient 2 Gleason 7 tumor and Patient 3 Gleason 7 tumorrespectively. These results provide evidence that 84P2A9 is a verytestis specific gene that is up-regulated in prostate cancer andpotentially other cancers. Similar to the MAGE antigens, 84P2A9 may thusqualify as a cancer-testis antigen (Van den Bynde and Boon, Int J ClinLab Res. 27:81-86, 1997).

FIG. 7 shows RNA was isolated from kidney cancers (1) and their adjacentnormal tissues (N) obtained from kidney cancer patients. Lanes 1-15 show769-P— clear cell type; A498—clear cell type; SW839—clear cell type;Normal Kidney; Patient 1, N; Patient 1, tumor; Patient 2, N; Patient 2,tumor, clear cell type, grade III; Patient 3, N; Patient 3, tumor,—clearcell type, grade II/IV; Patient 4, N; Patient 4, tumor, clear cell type,grade II/IV; Patient 5, N; Patient 5, tumor, clear cell type, grade II;and Patient 6, tumor, metastasis to chest wall respectively (N=normaladjacent tissue and CL=cell line). Northern analysis was performed using10 μg of total RNA for each sample. Expression of 84P2A9 was seen in all6 tumor samples tested as well as in the three kidney cell lines, 769-P,A498 and SW839.

FIG. 8 shows RNA was isolated from colon cancers (1) and their adjacentnormal tissues (N) obtained from colon cancer patients. Lanes 1-11 showColo 205; LoVo; T84; Caco-2; Patient 1, N; Patient 1, tumor, grade 2,T3N1Mx (positive for lymph node metastasis); Patient 2, N; Patient 2,tumor, grade 1, T2N0Mx; Patient 3, N; Patient 3, tumor, grade 1, T2N1Mx(positive for lymph node metastasis); and Patient 4, tumor, grade 2, T3N1 MX (positive for lymph node metastasis); respectively (N=normaladjacent tissue and CL=cell line). Northern analysis was performed using10 μg of total RNA for each sample. Expression of 84P2A9 was seen in all4 tumor samples tested as well as in the 4 colon cancer cell lines Colo205, LoVo, T84 and Caco-2.

FIG. 9 Shows expression of 84P2A9 assayed in a panel of human cancers(T) and their respective matched normal tissues (N) on RNA dot blots.Cancer cell lines from left to right are HeLa (cervical carcinoma),Daudi (Burkitt's lymphoma), K562 (CML), HL-60 (PML), G361 (melanoma),A549 (lung carcinoma), MOLT4 (lymphoblastic leuk.), SW480 (colorectalcarcinoma) and Raji (Burkitt's lymphoma). 84P2A9 expression was seen inkidney cancers, breast cancers, prostate cancers, lung cancers, stomachcancers, colon cancers, cervical cancers and rectum cancers. 84P2A9 wasalso found to be highly expressed in a panel of cancer cell lines,specially the MOLT-4 lymphoblastic leukemia and the A549 lung carcinomacell lines. The expression detected in normal adjacent tissues (isolatedfrom diseased tissues) but not in normal tissues, isolated from healthydonors, can indicate that these tissues are not fully normal and that84P2A9 can be expressed in early stage tumors.

FIG. 10 shows the expression of 84P2A9 in bladder cancer patientspecimens. Expression of 84P2A9 was seen in 4 bladder cancer patientspecimens tested and in three bladder cell lines (CL), UM-UC-3 (lane 1),J82 (lane 2) and SCABER (lane 3). RNA was isolated from normal bladder(Nb), bladder tumors (I) and their adjacent normal tissues (N) obtainedfrom 6 bladder cancer patients (P). Tumor from P1 is transitionalcarcinoma, grade 4; P2 is invasive squamous carcinoma; P3 istransitional carcinoma, grade 3; P4 is non-invasive papillary carcinoma,grade 1/3; P5 is papillary carcinoma, grade 3/3; and P6 is transitionalcarcinoma, grade 3/2. Northern analysis was performed using 10 μg oftotal RNA for each sample.

FIG. 11 shows the expression of 84P2A9 protein in 293T cells. 293T cellswere transiently transfected with either pcDNA3.1 V5-HIS epitope tagged84P2A9 plasmid or with empty control vector and harvested 2 days later.Cells were lysed in SDS-PAGE sample buffer and lysates were separated ona 10-20% SDS-PAGE gel and then transferred to nitrocellulose. The blotwas blocked in Tris-buffered saline (TBS)+2% non-fat milk and thenprobed with a 1:3,000 dilution of murine anti-V5 monoclonal Ab(Invitrogen) in TBS+0.15% Tween-20.+−.1% milk. The blot was washed andthen incubated with a 1:4,000 dilution of anti-mouse IgG-HRP conjugatesecondary antibody. Following washing, anti-V5 epitope immunoreactivebands were developed by enhanced chemiluminescence and visualized byexposure to autoradiographic film. Indicated by arrow is a specificanti-V5 immunoreactive band of approximately 87 Kd that corresponds toexpression of the epitope-tagged 84P2A9 protein in the transfectedcells.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe—inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are generally well understood and commonly employedusing conventional methodology by those skilled in the art, such as, forexample, the widely utilized molecular cloning methodologies describedin Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Asappropriate, procedures involving the use of commercially available kitsand reagents are generally carried out in accordance with manufacturerdefined protocols and/or parameters unless otherwise noted.

DEFINITIONS

As used herein, the terms “advanced prostate cancer”, “locally advancedprostate cancer”, “advanced disease” and “locally advanced disease” meanprostate cancers that have extended through the prostate capsule, andare meant to include stage C disease under the American UrologicalAssociation (AUA) system, stage C1-C2 disease under the Whitmore-jewettsystem, and stage T3-T4 and N+ disease under the TNM (tumor, node,metastasis) system. In general, surgery is not recommended for patientswith locally advanced disease, and these patients have substantiallyless favorable outcomes compared to patients having clinically localized(organ-confined) prostate cancer. Locally advanced disease is clinicallyidentified by palpable evidence of induration beyond the lateral borderof the prostate, or asymmetry or induration above the prostate base.Locally advanced prostate cancer is presently diagnosed pathologicallyfollowing radical prostatectomy if the tumor invades or penetrates theprostatic capsule, extends into the surgical margin, or invades theseminal vesicles.

The term “antibody” is used in the broadest sense. Therefore an“antibody” can be naturally occurring or man made such as monoclonalantibodies produced by conventional hybridoma technology. Anti-84P2A9antibodies comprise monoclonal and polyclonal antibodies as well asfragments containing the antigen binding domain and/or one or morecomplementarity determining regions of these antibodies. As used herein,an antibody fragment is defined as at least a portion of the variableregion of the immunoglobulin molecule that binds to its target, i.e.,the antigen binding region. In one embodiment it specifically coverssingle anti-84P2A9 antibody (including agonist, antagonist andneutralizing antibodies) and anti-84P2A9 antibody compositions withpolyepitopic specificity. The term “monoclonal antibody” as used hereinrefers to an antibody obtained from a population of substantiallyhomogeneous antibodies, i.e., the antibodies comprising the populationare identical except for possible naturally-occurring mutations that arepresent in minor amounts.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents, and toxins such as smallmolecule toxins or enzymatically active toxins of bacterial, fungalplant or animal origin, including fragments and/or variants thereof.

As used herein, the terms “hybridize”, “hybridizing”, “hybridizes” andthe like, used in the context of polynucleotides, are meant to refer toconventional hybridization conditions, preferably such as hybridizationin 50% formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperaturesfor hybridization are above 37° C. and temperatures for washing in0.1×SSC/0.1% SDS are above 55° C.

As used herein, a polynucleotide is said to be “isolated” when it issubstantially separated from contaminant polynucleotides that correspondor are complementary to genes other than the 84P2A9 gene or that encodepolypeptides other than 84P2A9 gene product or fragments thereof. Askilled artisan can readily employ nucleic acid isolation procedures toobtain an isolated 84P2A9 polynucleotide.

As used herein, a protein is said to be “isolated” when physical,mechanical or chemical methods are employed to remove the 84P2A9 proteinfrom cellular constituents that are normally associated with theprotein. A skilled artisan can readily employ standard purificationmethods to obtain an isolated 84P2A9 protein.

The term “mammal” as used herein refers to any mammal classified as amammal, including mice, rats, rabbits, dogs, cats, cows, horses andhumans. In one preferred embodiment of the invention, the mammal is amouse. In another preferred embodiment of the invention, the mammal is ahuman.

As used herein, the terms “metastatic prostate cancer” and “metastaticdisease” mean prostate cancers that have spread to regional lymph nodesor to distant sites, and are meant to include stage D disease under theAUA system and stage TxNxM+ under the TNM system. As is the case withlocally advanced prostate cancer, surgery is generally not indicated forpatients with metastatic disease, and hormonal (androgen ablation)therapy is a preferred treatment modality. Patients with metastaticprostate cancer eventually develop an androgen-refractory state within12 to 18 months of treatment initiation, and approximately half of thesepatients die within 6 months after developing androgen refractorystatus. The most common site for prostate cancer metastasis is bone.Prostate cancer bone metastases are often characteristicallyosteoblastic rather than osteolytic (i.e., resulting in net boneformation). Bone metastases are found most frequently in the spine,followed by the femur, pelvis, rib cage, skull and humerus. Other commonsites for metastasis include lymph nodes, lung, liver and brain.Metastatic prostate cancer is typically diagnosed by open orlaparoscopic pelvic lymphadenectomy, whole body radionuclide scans,skeletal radiography, and/or bone lesion biopsy.

“Moderately stringent conditions” are described by, identified but notlimited to, those in Sambrook et al., Molecular Cloning: A LaboratoryManual, New York: Cold Spring Harbor Press, 1989, and include the use ofwashing solution and hybridization conditions (e.g., temperature, ionicstrength and % SDS) less stringent than those described above. Anexample of moderately stringent conditions is overnight incubation at37° C. in a solution comprising; 20% formamide, 5×SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmonsperm DNA, followed by washing the filters in 1×SSC at about 37-50° C.The skilled artisan will recognize how to adjust the temperature, ionicstrength, etc. as necessary to accommodate factors such as probe lengthand the like.

As used herein “motif” as in biological motif of an 84P2A9-relatedprotein, refers to any set of amino acids forming part of the primarysequence of a protein, either contiguous or capable of being aligned tocertain positions that are generally invariant or conserved, that isassociated with a particular function or modification (e.g. that isphosphorylated, glycosylated or amidated).

As used herein, the term “polynucleotide” means a polymeric form ofnucleotides of at least 10 bases or base pairs in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide, and is meant to include single and double stranded forms ofDNA and/or RNA. In the art, this term if often used interchangeably with“oligonucleotide”. As discussed herein, an polynucleotide can comprise anucleotide sequence disclosed herein wherein thymidine (T) (as shown forexample in SEQ ID NO: 1) can also be uracil (U). This descriptionpertains to the differences between the chemical structures of DNA andRNA, in particular the observation that one of the four major bases inRNA is uracil (U) instead of thymidine (I).

As used herein, the term “polypeptide” means a polymer of at least about4, 5, 6, 7, or 8 amino acids. Throughout the specification, standardthree letter or single letter designations for amino acids are used. Inthe art, this term if often used interchangeably with “peptide”.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured nucleic acidsequences to reanneal when complementary strands are present in anenvironment below their melting temperature. The higher the degree ofdesired homology between the probe and hybridizable sequence, the higherthe relative temperature that can be used. As a result, it follows thathigher relative temperatures would tend to make the reaction conditionsmore stringent, while lower temperatures less so. For additional detailsand explanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, are identified by, but not limited to, those that (1) employ lowionic strength and high temperature for washing, for example 0.015 Msodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at50° C.; (2) employ during hybridization a denaturing agent, such asformamide, for example, 50% (v/v) formamide with 0.1% bovine serumalbumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodiumcitrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate,5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS,and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC(sodium chloride/sodium. citrate) and 50% formamide at 55° C., followedby a high-stringency wash consisting of 0.1×SSC containing EDTA at 55°C.

A “transgenic animal” (e.g., a mouse or rat) is an animal having cellsthat contain a transgene, which transgene was introduced into the animalor an ancestor of the animal at a prenatal e.g., an embryonic stage. A“transgene” is a DNA that is integrated into the genome of a cell fromwhich a transgenic animal develops.

As used herein, the 84P2A9 gene and protein is meant to include the84P2A9 genes and proteins specifically described herein and the genesand proteins corresponding to other 84P2A9 encoded proteins or peptidesand structurally similar variants of the foregoing. Such other 84P2A9peptides and variants will generally have coding sequences that arehighly homologous to the 84P2A9 coding sequence, and preferably share atleast about 50% amino acid homology (using BLAST criteria) andpreferably 50%, 60%, 70%, 80%, 90% or more nucleic acid homology, and atleast about 60% amino acid homology (using BLAST criteria), morepreferably sharing 70% or greater homology (using BLAST criteria).

The 84P2A9-related proteins of the invention include those specificallyidentified herein, as well as allelic variants, conservativesubstitution variants and homologs that can be isolated/generated andcharacterized without undue experimentation following the methodsoutlined herein or are readily available in the art Fusion proteins thatcombine parts of different 84P2A9 proteins or fragments thereof, as wellas fusion proteins of an 84P2A9 protein and a heterologous polypeptideare also included. Such 84P2A9 proteins are collectively referred to asthe 84P2A9-related proteins, the proteins of the invention, or 84P2A9.As used herein, the term “84P2A9-related polypeptide” refers to apolypeptide fragment or an 84P2A9 protein sequence of 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15 or more ammo acids

Structure and Expression of 84P2A9

As discussed in detail below, experiments with the LAPC-4 AD xenograftin male SCID mice have resulted in the identification of genes that areinvolved in the progression of androgen dependent (AD) prostate cancerto androgen independent (AI) cancer. Briefly, mice that harbored LAPC-4AD xenografts were castrated when the tumors reached a size of 1 cm indiameter. The tumors regressed in size and temporarily stopped producingthe androgen dependent protein PSA. Seven to fourteen dayspost-castration, PSA levels were detectable again in the blood of themice. Eventually such tumors develop an AI phenotype and start growingagain in the castrated males. Tumors were harvested at different timepoints after castration to identify genes that are turned on or offduring the transition to androgen independence.

Suppression subtractive hybridization (SSH) (Diatchenko et al., 1996,PNAS 93:6025) was then used to identify novel genes, such as those thatare overexpressed in prostate cancer, by comparing cDNAs from variousandrogen dependent and androgen independent LAPC xenografts. Thisstrategy resulted in the identification of novel genes exhibiting tissueand cancer specific expression. One of these genes, designated 84P2A9,was identified from a subtraction where cDNA derived from an LAPC-4 ADtumor, 3 days post-castration, was subtracted from cDNA derived from anLAPC-4 AD tumor grown in an intact male. The SSH DNA sequence of about425 bp (FIG. 1) is novel and exhibits homology only to expressedsequence tags (ESTs) in the dbEST database.

84P2A9, encodes a putative nuclear protein that exhibits prostate andtestis-related expression. The initial characterization of 84P2A9indicates that it is aberrantly expressed multiple cancers includingprostate, testis, kidney, brain, bone, skin, ovarian, breast, pancreas,colon, lymphocytic and lung cancers. The expression of 84P2A9 inprostate cancer provides evidence that this protein has a functionalrole in tumor progression. It is possible that 84P2A9 functions as atranscription factor involved in activating genes involved intumorigenesis or repressing genes that block tumorigenesis.

As is further described in the Examples that follow, the 84P2A9 genesand proteins have been characterized using a number of analyticalapproaches. For example, analyses of nucleotide coding and amino acidsequences were conducted in order to identify potentially relatedmolecules, as well as recognizable structural domains, topologicalfeatures, and other elements within the 84P2A9 mRNA and proteinstructures. Northern blot analyses of 84P2A9 mRNA expression wereconducted in order to establish the range of normal and canceroustissues expressing 84P2A9 message.

A full length 84P2A9 cDNA clone (clone 1) of 2345 base pairs (SEQ IDNO: 1) was cloned from an LAPC-4 AD cDNA library (Lambda ZAP Express,Stratagene) (FIG. 2). The cDNA encodes an open reading frame (ORF) of504 amino acids (SEQ ID NO: 2). Sequence analysis revealed the presenceof six potential nuclear localization signals and is predicted to benuclear using the PSORT program. The protein sequence has some homologyto a human brain protein KIAA1152 (SEQ ID NO: 5) (39.5% identity over a337 amino acid region), and contains a domain that is homologous to theLUCA15 tumor suppressor protein (SEQ ID NO: 6) (64.3% identity over a 42amino acid region) (GenBank Accession #P52756) (FIG. 3).

84P2A9 expression is prostate and testis-related in normal adult humantissues, but is also expressed in certain cancers, including prostate,testis, kidney, brain, bone, skin, ovarian, breast, pancreas, colon,lymphocytic and lung cancers. (see, e.g., FIGS. 4-8). Human prostatetumor xenografts originally derived from a patient with high grademetastatic prostate cancer express high levels of 84P2A9 (FIG. 4).

As disclosed herein, 84P2A9 exhibits specific properties that areanalogous to those found in a family of genes whose polynucleotides,polypeptides, reactive cytotoxic T cells (CTL), helper T cells (HTL) andanti-polypeptide antibodies are used in well known diagnostic assaysdirected to examining conditions associated with disregulated cellgrowth such as cancer, in particular prostate cancer (see, e.g., bothits highly specific pattern of tissue expression as well as itsoverexpression in prostate cancers as described for example in Example3). The best known member of this class is PSA, the archetypal markerthat has been used by medical practitioners for years to identify andmonitor the presence of prostate cancer (see, e.g., Merrill et al., J.Urol. 163(2): 503-5120 (2000); Polascik et al., J. Urol. August;162(2):293-306 (1999) and Fortier et al., J. Nat. Cancer Inst. 91(19):1635-1640(1999)). A variety of other diagnostic markers are also used inthis context including p53 and K-ras (see, e.g., Tulchinsky et al., IntJ Mol Med 1999 July; 4(1):99-102 and Minimoto et al., Cancer Detect Prev2000; 24(1):1-12). Therefore, this disclosure of the 84P2A9polynucleotides and polypeptides (as well as the 84P2A9 polynucleotideprobes and anti-84P2A9 antibodies used to identify the presence of thesemolecules) and their properties allows skilled artisans to utilize thesemolecules in methods that are analogous to those used, for example, in avariety of diagnostic assays directed to examining conditions associatedwith cancer.

Typical embodiments of diagnostic methods which utilize the 84P2A9polynucleotides, polypeptides and antibodies described herein areanalogous to those methods from well established diagnostic assays whichemploy PSA polynucleotides, polypeptides and antibodies. For example,just as PSA polynucleotides are used as probes (for example in Northernanalysis, see, e.g., Sharief et al., Biochem. Mol. Biol. Int.33(3):567-74(1994)) and primers (for example in PCR analysis, see, e.g.,Okegawa et al., J. Urol. 163(4): 1189-1190 (2000)) to observe thepresence and/or the level of PSA mRNAs in methods of monitoring PSAoverexpression or the metastasis of prostate cancers, the 84P2A9polynucleotides described herein can be utilized in the same way todetect 84P2A9 overexpression or the metastasis of prostate and othercancers expressing this gene. Alternatively, just as PSA polypeptidesare used to generate antibodies specific for PSA which can then be usedto observe the presence and/or the level of PSA proteins in methods ofmonitoring PSA protein overexpression (see, e.g., Stephan et al.,Urology 55(4):560-3 (2000)) or the metastasis of prostate cells (see,e.g., Alanen et al., Pathol. Res. Pract. 192(3):233-7 (1996)), the84P2A9 polypeptides described herein can be utilized to generateantibodies for use in detecting 84P2A9 overexpression or the metastasisof prostate cells and cells of other cancers expressing this gene.

Specifically, because metastases involves the movement of cancer cellsfrom an organ of origin (such as the testis or prostate gland etc.) to adifferent area of the body (such as a lymph node), assays which examinea biological sample for the presence of cells expressing 84P2A9polynucleotides and/or polypeptides can be used to provide evidence ofmetastasis. For example, when a biological sample from tissue that doesnot normally contain 84P2A9 expressing cells (lymph node) is found tocontain 84P2A9 expressing cells such as the 84P2A9 expression seen inLAPC4 and LAPC9, xenografts isolated from lymph node and bonemetastasis, respectively, this finding is indicative of metastasis.

Alternatively 84P2A9 polynucleotides and/or polypeptides can be used toprovide evidence of cancer, for example, when a cells in biologicalsample that do not normally express 84P2A9 or express 84P2A9 at adifferent level are found to express 84P2A9 or have an increasedexpression of 84P2A9 (see, e.g., the 84P2A9 expression in kidney, lungand colon cancer cells and in patient samples etc. shown in FIGS. 4-10).In such assays, artisans may father wish to generate supplementaryevidence of metastasis by testing the biological sample for the presenceof a second tissue restricted marker (in addition to 84P2A9) such asPSA, PSCA etc. (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3):233-237 (1996)).

Just as PSA polynucleotide fragments and polynucleotide variants areemployed by skilled artisans for use in methods of monitoring PSA,84P2A9 polynucleotide fragments and polynucleotide variants are used inan analogous manner. In particular, typical PSA polynucleotides used inmethods of monitoring PSA are probes or primers which consist offragments of the PSA cDNA sequence. Illustrating this, primers used toPCR amplify a PSA polynucleotide must include less than the whole PSAsequence to function in the polymerase chain reaction. In the context ofsuch PCR reactions, skilled artisans generally create a variety ofdifferent polynucleotide fragments that can be used as primers in orderto amplify different portions of a polynucleotide of interest or tooptimize amplification reactions (see, e.g., Caetano-Anolles, G.Biotechniques 25(3): 472-476, 478-480 (1998); Robertson et al., MethodsMol. Biol. 98:121-154 (1998)). An additional illustration of the use ofsuch fragments is provided in Example 3, where an 84P2A9 polynucleotidefragment is used as a probe to show the overexpression of 84P2A9 mRNAsin cancer cells. In addition, in order to facilitate their use bymedical practitioners, variant polynucleotide sequences are typicallyused as primers and probes for the corresponding mRNAs in PCR andNorthern analyses (see, e.g., Sawai et al., Fetal Diagn. Ther. 1996November-December; 11(6):407-13 and Current Protocols In MolecularBiology, Volume 2, Unit 2, Frederick M. Ausubul et al. eds., 1995)).Polynucleotide fragments and variants are typically useful in thiscontext as long as they have the common attribute or characteristic ofbeing capable of binding to a target polynucleotide sequence (e.g. the84P2A9 polynucleotide shown in SEQ ID NO: 1) under conditions of highstringency.

Just as PSA polypeptide fragments and polypeptide variants are employedby skilled artisans for use in methods of monitoring the PSA molecule,84P2A9 polypeptide fragments and polypeptide variants can also be usedin an analogous manner. In particular, typical PSA polypeptides used inmethods of monitoring PSA are fragments of the PSA protein which containan antibody epitope that can be recognized by an antibody or T cell thatspecifically binds to the PSA protein. This practice of usingpolypeptide fragments or polypeptide variants to generate antibodies(such as anti-PSA antibodies or T cells) is typical in the art with awide variety of systems such as fusion proteins being used bypractitioners (see, e.g., Current Protocols In Molecular Biology, Volume2, Unit 16, Frederick M. Ausubul et al. eds., 1995). In this context,each epitope(s) in a protein of interest functions to provide thearchitecture with which an antibody or T cell is reactive. Typically,skilled artisans generally create a variety of different polypeptidefragments that can be used in order to generate antibodies specific fordifferent portions of a polypeptide of interest (see, e.g., U.S. Pat.No. 5,840,501 and U.S. Pat. No. 5,939,533). For example it may bepreferable to utilize a polypeptide comprising one of the 84P2A9biological motifs discussed herein or available in the art. Polypeptidefragments and variants or analogs are typically useful in this contextas long as they comprise an epitope capable of generating an antibody orT cell specific for a target polypeptide sequence (e.g. the 84P2A9polypeptide shown in SEQ ID NO: 2).

As shown herein, the 84P2A9 polynucleotides and polypeptides (as well asthe 84P2A9 polynucleotide probes and anti-84P2A9 antibodies or T cellsused to identify the presence of these molecules) exhibit specificproperties that make them useful in diagnosing cancers of the prostate.Diagnostic assays that measure the presence of 84P2A9 gene products, inorder to evaluate the presence or onset of the particular diseaseconditions described herein such as prostate cancer are particularlyuseful in identifying patients for preventive measures or furthermonitoring, as has been done so successfully with PSA. Moreover, thesematerials satisfy a need in the art for molecules having similar orcomplementary characteristics to PSA in situations where, for example, adefinite diagnosis of metastasis of prostatic origin cannot be made onthe basis of a testing for PSA alone (see, e.g., Alanen et al., Pathol.Res. Pract. 192(3): 233-237 (1996)), and consequently, materials such as84P2A9 polynucleotides and polypeptides (as well as the 84P2A9polynucleotide probes and anti-84P2A9 antibodies used to identify thepresence of these molecules) must be employed to confirm metastases ofprostatic origin.

Finally, in addition to their use in diagnostic assays, the 84P2A9polynucleotides disclosed herein have a number of other specificutilities such as their use in the identification of oncogeneticassociated chromosomal abnormalities in 1q32.3. Moreover, in addition totheir use in diagnostic assays, the 84P2A9-related proteins andpolynucleotides disclosed herein have other utilities such as their usein the forensic analysis of tissues of unknown origin (see, e.g.,Takahama K Forensic Sci Int 1996 Jun. 28; 80(1-2): 63-9).

84P2A9 Polynucleotides

One aspect of the invention provides polynucleotides corresponding orcomplementary to all or part of an 84P2A9 gene, mRNA, and/or codingsequence, preferably in isolated form, including polynucleotidesencoding an 84P2A9 protein and fragments thereof, DNA, RNA, DNA/RNAhybrid, and related molecules, polynucleotides or oligonucleotidescomplementary to an 84P2A9 gene or mRNA sequence or a part thereof, andpolynucleotides or oligonucleotides that hybridize to an 84P2A9 gene,mRNA, or to an 84P2A9 encoding polynucleotide (collectively, “84P2A9polynucleotides”).

One embodiment of an 84P2A9 polynucleotide is an 84P2A9 polynucleotidehaving the sequence shown in SEQ ID NO: 1. An 84P2A9 polynucleotide cancomprise a polynucleotide having the nucleotide sequence of human 84P2A9as shown in SEQ ID NO: 1, wherein T can also be U; a polynucleotide thatencodes all or part of the 84P2A9 protein; a sequence complementary tothe foregoing; or a polynucleotide fragment of any of the foregoing.Another embodiment comprises a polynucleotide having the sequence asshown in SEQ ID NO: 1, from nucleotide residue number 163 throughnucleotide residue number 1674, or from residue number 718 throughresidue number 1390, wherein T can also be U. Another embodimentcomprises a polynucleotide encoding an 84P2A9 polypeptide whose sequenceis encoded by the cDNA contained in the plasmid as deposited withAmerican Type Culture Collection as Accession No. PTA-1151 Anotherembodiment comprises a polynucleotide that is capable of hybridizingunder stringent hybridization conditions to the human 84P2A9 cDNA shownin SEQ ID NO: 1 or to a polynucleotide fragment thereof.

Typical embodiments of the invention disclosed herein include 84P2A9polynucleotides encoding specific portions of the 84P2A9 mRNA sequence(and those which are complementary to such sequences) such as those thatencode the protein and fragments thereof, for example of 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. For example,representative embodiments of the invention disclosed herein include:polynucleotides encoding about amino acid position 1 to about amino acid10 of the 84P2A9 protein shown in FIG. 2 (SEQ ID NO: 2), polynucleotidesencoding about amino acid 10 to about amino acid 20 of the 84P2A9protein shown in FIG. 2, polynucleotides encoding about amino acid 20 toabout amino acid 30 of the 84P2A9 protein shown in FIG. 2,polynucleotides encoding about amino acid 30 to about amino acid 40 ofthe 84P2A9 protein shown in FIG. 2, polynucleotides encoding about aminoacid 40 to about amino acid 50 of the 84P2A9 protein shown in FIG. 2,polynucleotides encoding about amino acid 50 to about amino acid. 60 ofthe 84P2A9 protein shown in FIG. 2, polynucleotides encoding about aminoacid 60 to about amino acid 70 of the 84P2A9 protein shown in FIG. 2,polynucleotides encoding about amino acid 70 to about amino acid 80 ofthe 84P2A9 protein shown in FIG. 2, polynucleotides encoding about aminoacid 80 to about amino acid 90 of the 84P2A9 protein shown in FIG. 2 andpolynucleotides encoding about amino acid 90 to about amino acid 100 ofthe 84P2A9 protein shown in FIG. 2, etc. Following this scheme,polynucleotides (of at least 10 nucleic acids) encoding portions of theamino acid sequence of amino acids 100-504 of the 84P2A9 protein aretypical embodiments of the invention.

Polynucleotides encoding larger portions of the 84P2A9 protein are alsocontemplated. For example polynucleotides encoding from about amino acid1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 30, or 40 or 50etc.) of the 84P2A9 protein shown in FIG. 2 can be generated by avariety of techniques well known in the art. An illustrative embodimentof such a polynucleotide consists of a polynucleotide having thesequence as shown in FIG. 2, from nucleotide residue number 718 throughnucleotide residue number 1390.

Additional illustrative embodiments of the invention disclosed hereininclude 84P2A9 polynucleotide fragments encoding one or more of thebiological motifs contained within the 84P2A9 protein sequence. In oneembodiment, typical polynucleotide fragments of the invention can encodeone or more of the nuclear localization sequences disclosed herein. Inanother embodiment, typical polynucleotide fragments of the inventioncan encode one or more of the regions of 84P2A9 that exhibit homology toLUCA 15 and/or KIAA1152 and/or NY-Lu-12 lung cancer antigen (AF 042857),which exhibits Zinc finger and RNA binding motifs (see, e.g., Gure etal., Cancer Res. 58(5): 1034-1041 (1998). In another embodiment of theinvention, typical polynucleotide fragments can encode one or more ofthe 84P2A9 N-glycosylation sites, cAMP and cCMP-dependent protein kinasephosphorylation sites, casein kinase II phosphorylation sites orN-myristoylation site and amidation sites as disclosed in greater detailin the text discussing the 84P2A9 protein and polypeptides herein. Inyet another embodiment of the invention, typical polynucleotidefragments can encode sequences that are unique to one or more 84P2A9alternative splicing variants, such as the splice variant that generatesthe 4.5 KB transcript that is overexpressed in prostate cancers shown inFIG. 4.

The polynucleotides of the preceding paragraphs have a number ofdifferent specific uses. For example, because the human 84P2A9 gene mapsto chromosome 1q32.3, polynucleotides encoding different regions of the84P2A9 protein can be used to characterize cytogenetic abnormalities onchromosome 1, band q32 that have been identified as being associatedwith various cancers. In particular, a variety of chromosomalabnormalities in 1 q32 including translocations and deletions have beenidentified as frequent cytogenetic abnormalities in a number ofdifferent cancers (see, e.g., Bieche et al., Genes Chromosomes Cancer,24(3): 255-263 (1999); Gorunova et al., Genes Chromosomes Cancer, 26(4):312-321 (1999); Reid et al., Cancer Res. (22): 5415-5423 (1995)).Consequently, polynucleotides encoding specific regions of the 84P2A9protein provide new tools that can be used to delineate with a greaterprecision than previously possible, the specific nature of thecytogenetic abnormalities in this region of chromosome 1 that cancontribute to the malignant phenotype. In this context, thesepolynucleotides satisfy a need in the art for expanding the sensitivityof chromosomal screening in order to identify more subtle and lesscommon chromosomal abnormalities (see, e.g., Evans et al., Am. J.Obstet. Gynecol 171(4): 1055-1057 (1994)).

Alternatively, as 84P2A9 is shown to be highly expressed in prostatecancers (FIG. 4), these polynucleotides can be used in methods assessingthe status of 84P2A9 gene products in normal versus cancerous tissues.Typically, polynucleotides encoding specific regions of the 84P2A9protein can be used to assess the presence of perturbations (such asdeletions, insertions, point mutations, or alterations resulting in aloss of an antigen etc.) in specific regions (such regions containing anuclear localization signal) of the 84P2A9 gene products. Exemplaryassays include both RT-PCR assays as well as single-strand conformationpolymorphism (SSCP) analysis (see, e.g., Marrogi et al., J. Cutan.Pathol. 26(8): 369-378 (1999), both of which utilize polynucleotidesencoding specific regions of a protein to examine these regions withinthe protein.

Other specifically contemplated nucleic acid related embodiments of theinvention disclosed herein are genomic DNA, cDNAs, ribozymes, andantisense molecules, as well as nucleic acid molecules based on analternative backbone or including alternative bases, whether derivedfrom natural sources or synthesized. For example, antisense moleculescan be RNAs or other molecules, including peptide nucleic acids (PNAs)or non-nucleic acid molecules such as phosphorothioate derivatives, thatspecifically bind DNA or RNA in a base pair-dependent manner. A skilledartisan can readily obtain these classes of nucleic acid molecules usingthe 84P2A9 polynucleotides and polynucleotide sequences disclosedherein.

Antisense technology entails the administration of exogenousoligonucleotides that bind to a target polynucleotide located within thecells. The term “antisense” refers to the fact that sucholigonucleotides are complementary to their intracellular targets, e.g.,84P2A9. See for example, Jack Cohen, OLIGODEOXYNUCLEOTIDES, AntisenseInhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5(1988). The 84P2A9 antisense oligonucleotides of the present inventioninclude derivatives such as S-oligonucleotides (phosphorothioatederivatives or S-oligos, see, Jack Cohen, supra), which exhibit enhancedcancer cell growth inhibitory action. S-oligos (nucleosidephosphorothioates) are isoelectronic analogs of an oligonucleotide(O-oligo) in which a nonbridging oxygen atom of the phosphate group isreplaced by a sulfur atom. The S-oligos of the present invention can beprepared by treatment of the corresponding O-oligos with3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transferreagent See Iyer, R. P. et al, J. Org. Chem. 55:4693-4698 (1990); andIyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254 (1990). Additional84P2A9 antisense oligonucleotides of the present invention includemorpholino antisense oligonucleotides known in the art (see, e.g.,Partridge et al., 1996, Antisense & Nucleic Acid Drug Development 6:169-175).

The 84P2A9 antisense oligonucleotides of the present invention typicallycan be RNA or DNA that is complementary to and stably hybridizes withthe first 100 N-terminal codons or last 100 C-terminal codons of the84P2A9 genomic sequence or the corresponding mRNA. Absolutecomplementarity is not required, although high degrees ofcomplementarity are preferred. Use of an oligonucleotide complementaryto this region allows for the selective hybridization to 84P2A9 mRNA andnot to mRNA specifying other regulatory subunits of protein kinase.Preferably, the 84P2A9 antisense oligonucleotides of the presentinvention are a 15 to 30-mer fragment of the antisense DNA moleculehaving a sequence that hybridizes to 84P2A9 mRNA. Optionally, 84P2A9antisense oligonucleotide is a 30-mer oligonucleotide that iscomplementary to a region in the first 10 N-terminal codons or last 10C-terminal codons of 84P2A9. Alternatively, the antisense molecules aremodified to employ ribozymes in the inhibition of 84P2A9 expression. L.A. Couture & D. T. Stinchcomb; Tends Genet 12: 510-515 (1996).

Further specific embodiments of this aspect of the invention includeprimers and primer pairs, which allow the specific amplification of thepolynucleotides of the invention or of any specific parts thereof, andprobes that selectively or specifically hybridize to nucleic acidmolecules of the invention or to any part thereof. Probes can be labeledwith a detectable marker, such as, for example, a radioisotope,fluorescent compound, bioluminescent compound, a chemiluminescentcompound, metal chelator or enzyme. Such probes and primers can be usedto detect the presence of an 84P2A9 polynucleotide in a sample and as ameans for detecting a cell expressing an 84P2A9 protein.

Examples of such probes include polypeptides comprising all or part ofthe human 84P2A9 cDNA sequences shown in FIG. 2. Examples of primerpairs capable of specifically amplifying 84P2A9 mRNAs are also describedin the Examples that follow. As will be understood by the skilledartisan, a great many different primers and probes can be prepared basedon the sequences provided herein and used effectively to amplify and/ordetect an 84P2A9 mRNA.

The 84P2A9 polynucleotides of the invention are useful for a variety ofpurposes, including but not limited to their use as probes and primersfor the amplification and/or detection of the 84P2A9 gene(s), mRNA(s),or fragments thereof; as reagents for the diagnosis and/or prognosis ofprostate cancer and other cancers; as coding sequences capable ofdirecting the expression of 84P2A9 polypeptides; as tools for modulatingor inhibiting the expression of the 84P2A9 gene(s) and/or translation ofthe 84P2A9 transcript(s); and as therapeutic agents.

Isolation of 84P2A9-Encoding Nucleic Acid Molecules

The 84P2A9 cDNA sequences described herein enable the isolation of otherpolynucleotides encoding 84P2A9 gene product(s), as well as theisolation of polynucleotides encoding 84P2A9 gene product homologs,alternatively spliced isoforms, allelic variants, and mutant forms ofthe 84P2A9 gene product Various molecular cloning methods that can beemployed to isolate full length cDNAs encoding an 84P2A9 gene are wellknown (See, for example, Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, 2d edition., Cold Spring Harbor Press, New York,1989; Current Protocols in Molecular Biology. Ausubel et al., Eds.,Wiley and Sons, 1995). For example, lambda phage cloning methodologiescan be conveniently employed, using commercially available cloningsystems (e.g., Lambda ZAP Express, Stratagene). Phage clones containing84P2A9 gene cDNAs can be identified by probing with a labeled 84P2A9cDNA or a fragment thereof. For example, in one embodiment, the 84P2A9cDNA (FIG. 2) or a portion thereof can be synthesized and used as aprobe to retrieve overlapping and full length cDNAs corresponding to an84P2A9 gene. The 84P2A9 gene itself can be isolated by screening genomicDNA libraries, bacterial artificial chromosome libraries (BACs), yeastartificial chromosome libraries (YACs), and the like, with 84P2A9 DNAprobes or primers.

Recombinant DNA Molecules and Host-Vector Systems

The invention also provides recombinant DNA or RNA molecules containingan 84P2A9 polynucleotide or a fragment or analog or homologue thereof,including but not limited to phages, plasmids, phagemids, cosmids, YACs,BACs, as well as various viral and non-viral vectors well known in theart, and cells transformed or transfected with such recombinant DNA orRNA molecules. As used herein, a recombinant DNA or RNA molecule is aDNA or RNA molecule that has been subjected to molecular manipulation invitro. Methods for generating such molecules are well known (see, forexample, Sambrook et al, 1989, supra).

The invention further provides a host-vector system comprising arecombinant DNA molecule containing an 84P2A9 polynucleotide or fragmentor analog or homologue thereof within a suitable prokaryotic oreukaryotic host cell. Examples of suitable eukaryotic host cells includea yeast cell, a plant cell, or an animal cell, such as a mammalian cellor an insect cell (e.g., a baculovirus-infectible cell such as an Sf9 orHighFive cell). Examples of suitable mammalian cells include variousprostate cancer cell lines such as DU145 and TsuPr1, other transfectableor transducible prostate cancer cell lines, as well as a number ofmammalian cells routinely used for the expression of recombinantproteins (e.g., COS, CHO, 293, 293T cells). More particularly, apolynucleotide comprising the coding sequence of 84P2A9 or a fragment oranalog or homolog thereof can be used to generate 84P2A9 proteins orfragments thereof using any number of host-vector systems routinely usedand widely known in the art.

A wide range of host-vector systems suitable for the expression of84P2A9 proteins or fragments thereof are available, see for example,Sambrook et al., 1989, supra; Current Protocols in Molecular Biology,1995, supra). Preferred vectors for mammalian expression include but arenot limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviralvector pSR.alpha.tkneo (Muller et al., 1991, MCB 11: 1785). Using theseexpression vectors, 84P2A9 may be preferably expressed in severalprostate cancer and non-prostate cell lines, including for example 293,293T, rat-1, NIH 3T3 and TsuPr1. The host-vector systems of theinvention are useful for the production of an 84P2A9 protein or fragmentthereof. Such host-vector systems can be employed to study thefunctional properties of 84P2A9 and 84P2A9 mutations or analogs.

Recombinant human 84P2A9 protein or an analog or homolog or fragmentthereof can be produced by mammalian cells transfected with a constructencoding 84P2A9. In an illustrative embodiment described in theExamples, 293T cells can be transfected with an expression plasmidencoding 84P2A9 or fragment or analog or homolog thereof, the 84P2A9 orrelated protein is expressed in the 293T cells, and the recombinant84P2A9 protein can be isolated using standard purification methods(e.g., affinity purification using anti-84P2A9 antibodies). In anotherembodiment, also described in the Examples herein, the 84P2A9 codingsequence is subcloned into the retroviral vector pSR.alpha.MSVtkneo andused to infect various mammalian cell lines, such as NIH 3T3, TsuPr1,293 and rat-1 in order to establish 84P2A9 expressing cell lines.Various other expression systems well known in the art can also beemployed. Expression constructs encoding a leader peptide joined inframe to the 84P2A9 coding sequence can be used for the generation of asecreted form of recombinant 84P2A9 protein.

Proteins encoded by the 84P2A9 genes, or by analogs or homologs orfragments thereof, will have a variety of uses, including but notlimited to generating antibodies and in methods for identifying ligandsand other agents and cellular constituents that bind to an 84P2A9 geneproduct Antibodies raised against an 84P2A9 protein or fragment thereofcan be useful in diagnostic and prognostic assays, and imagingmethodologies in the management of human cancers characterized byexpression of 84P2A9 protein, including but not limited to cancers ofthe prostate and testis. Such antibodies can be expressedintracellularly and used in methods of treating patients with suchcancers. Various immunological assays useful for the detection of 84P2A9proteins are contemplated, including but not limited to various types ofradioimmunoassays, enzyme-linked immunosorbent assays (ELISA),enzyme-linked immunofluorescent assays (ELIFA), immunocytochemicalmethods, and the like. Such antibodies can be labeled and used asimmunological imaging reagents capable of detecting 84P2A9 expressingcells (e.g., in radioscintigraphic imaging methods). 84P2A9 proteins canalso be particularly useful in generating cancer vaccines, as furtherdescribed below.

84P2A9 Polypeptides

Another aspect of the present invention provides 84P2A9-related proteinsand polypeptide fragments thereof. Specific embodiments of 84P2A9proteins comprise a polypeptide having all or part of the amino acidsequence of human 84P2A9 as shown in FIG. 2. Alternatively, embodimentsof 84P2A9 proteins-comprise variant polypeptides having alterations inthe amino acid sequence of human 84P2A9 shown in FIG. 2.

In general, naturally occurring allelic variants of human 84P2A9 share ahigh degree of structural identity and homology (e.g., 90% or moreidentity). Typically, allelic variants of the 84P2A9-related proteinscontain conservative amino acid substitutions within the 84P2A9sequences described herein or contain a substitution of an amino acidfrom a corresponding position in a homologue of 84P2A9. One class of84P2A9 allelic variants are proteins that share a high degree ofhomology with at least a small region of a particular 84P2A9 amino acidsequence, but further contain a radical departure from the sequence,such as a non-conservative substitution, truncation, insertion or frameshift. In comparisons of protein sequences, the terms, Similarity,identity, and Homology each have a distinct meaning. Moreover, Orthologyand Paralogy are important concepts describing the relationship ofmembers of a given protein family in one organism to the members of thesame family in other organisms.

Conservative amino acid substitutions can frequently be made in aprotein without altering either the conformation or the function of theprotein. Such changes include substituting any of isoleucine (I), valine(V), and leucine (L) for any other of these hydrophobic amino acids;aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q)for asparagine (N) and vice versa; and serine (S) for threonine (T) andvice versa. Other substitutions can also be considered conservative,depending on the environment of the particular amino acid and its rolein the three-dimensional structure of the protein. For example, glycine(G) and alanine (A) can frequently be interchangeable, as can alanine(A) and valine (V). Methionine (M), which is relatively hydrophobic, canfrequently be interchanged with leucine and isoleucine, and sometimeswith valine. Lysine (K) and arginine (R) are frequently interchangeablein locations in which the significant feature of the amino acid residueis its charge and the differing pK's of these two amino acid residuesare not significant. Still other changes can be considered“conservative” in particular environments (see, e.g. Table 2 herein;pages 13-15 “Biochemistry” 2^(nd) ED. Lubert Stryer ed (StanfordUniversity); Henikoff et al., PNAS 1992 Vol 89 10915-10919; Lei et al.,J Biol Chem 1995 May 19; 270(20):11882-6).

Embodiments of the invention disclosed herein include a wide variety ofart accepted variants of 84P2A9 proteins such as polypeptides havingamino acid insertions, deletions and substitutions. 84P2A9 variants canbe made using methods known in the art such as site-directedmutagenesis, alanine scanning, and PCR mutagenesis. Site-directedmutagenesis [Carter et al., Nucl. Adds Res., 13:4331 (1986); Zoller etal., Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells etal., Gene, 34:315 (1985)], restriction selection mutagenesis [Wells etal., Philos. Trans. R. Soc. London SerA, 317:415 (1986)] or other knowntechniques can be performed on the cloned DNA to produce the 84P2A9variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence that is involved in aspecific biological activity such as a protein-protein interaction.Among the preferred scanning amino acids are relatively small, neutralamino acids. Such amino acids include alanine, glycine, serine, andcysteine. Alanine is typically a preferred scanning amino acid amongthis group because it eliminates the side-chain beyond the beta-carbonand is less likely to alter the main-chain conformation of the variant.Alanine is also typically preferred because it is the most common aminoacid. Further, it is frequently found in both buried and exposedpositions [Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia,J. Mol. Biol., 150:1 (1976)]. If alanine substitution does not yieldadequate amounts of variant, an isosteric amino acid can be used.

As defined herein, 84P2A9 variants, analogs or homologs, have thedistinguishing attribute of having at least one epitope in common withan 84P2A9 protein having the amino acid sequence of SEQ ID NO: 2, suchthat an antibody or T cell that specifically binds to an 84P2A9 variantwill also specifically bind to the 84P2A9 protein-having the amino acidsequence of SEQ ID NO: 2. A polypeptide ceases to be a variant of theprotein shown in SEQ ID NO: 2 when it no longer contains an epitopecapable of being recognized by an antibody or T cell that specificallybinds to an 84P2A9 protein. Those skilled in the art understand thatantibodies that recognize proteins bind to epitopes of varying size, anda grouping of the order of about four or five amino acids, contiguous ornot, is regarded as a typical number of amino acids in a minimalepitope. See, e.g., Nair et al., J. Immunol 2000 165(12): 6949-6955;Hebbes et al., Mol Immunol (1989) 26(9):865-73; Schwartz et al., JImmunol (1985) 135(4):2598-608. Another specific class of 84P2A9-relatedprotein variants shares 90% or more identity with the amino acidsequence of SEQ ID NO: 2 or a fragment thereof. Another specific classof 84P2A9 protein variants or analogs comprise one or more of the 84P2A9biological motifs described below or presently known in the art. Thus,encompassed by the present invention are analogs of 84P2A9 fragments(nucleic or amino acid) that altered functional (e.g. immunogenic)properties relative to the starting fragment. It is to be appreciatedthat motifs now or which become part of the art are to be applied to thenucleic or amino acid sequences of FIG. 2.

As discussed herein, embodiments of the claimed invention includepolypeptides containing less than the 504 amino acid sequence of the84P2A9 protein shown in FIG. 2. For example, representative embodimentsof the invention comprise peptides/proteins having any 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15 or more contiguous amino acids of the 84P2A9protein shown in FIG. 2 (SEQ ID NO: 2). Moreover, representativeembodiments of the invention disclosed herein include polypeptidesconsisting of about amino acid 1 to about amino acid 10 of the 84P2A9protein shown in FIG. 2, polypeptides consisting of about amino acid 10to about amino acid 20 of the 84P2A9 protein shown in FIG. 2,polypeptides consisting of about amino acid 20 to about amino acid 30 ofthe 84P2A9 protein shown in FIG. 2, polypeptides consisting of aboutamino acid 30 to about amino acid 40 of the 84P2A9 protein shown in FIG.2, polypeptides consisting of about amino acid 40 to about amino acid 50of the 84P2-A9 protein shown in FIG. 2, polypeptides consisting of aboutamino acid 50 to about amino acid 60 of the 84P2A9 protein shown in FIG.2, polypeptides consisting of about amino acid 60 to about amino acid 70of the 84P2A9 protein shown in FIG. 2, polypeptides consisting of aboutamino acid 70 to about amino acid 80 of the 84P2A9 protein shown in FIG.2, polypeptides consisting of about amino acid 80 to about amino acid 90of the 84P2A9 protein shown in FIG. 2 and polypeptides consisting ofabout amino acid 90 to about amino acid 100 of the 84P2A9 protein shownin FIG. 2, etc. throughout the entirety of the 84P2A9 sequence.Following this scheme, polypeptides consisting of portions of the aminoacid sequence of amino acids 100-504 of the 84P2A9 protein are typicalembodiments of the invention. Polypeptides consisting of larger portionsof the 84P2A9 protein are also contemplated. For example polypeptidesconsisting of about amino acid 1 (or 20 or 30 or 40 etc.) to about aminoacid 20, (or 30, or 40 or 50 etc.) of the 84P2A9 protein shown in FIG. 2can be generated by a variety of techniques well known in the art. It isto be appreciated that the starting and stopping positions in thisparagraph refer to the specified position as well as that position plusor minus 5 residues.

Additional illustrative embodiments of the invention disclosed hereininclude 84P2A9-related proteins containing the amino acid residues ofone or more of the biological motifs contained within the 84P2A9-relatedprotein sequence as shown in FIG. 2. In one embodiment, proteins of theinvention comprise one or more of the 84P2A9 nuclear localizationsequences such as RKRR at residues 4245 of SEQ ID NO: 2, RKRR atresidues 47-50 of SEQ ID NO: 2, KRRP at residues 101-104 of SEQ ID NO:2, RRRRRK at residues 135-139 of SEQ ID NO: 2 and/or KKRK at residues186-189 of SEQ ID NO: 2. In another embodiment, proteins of theinvention comprise one or more of the 84P2A9 N-glycosylation sites suchas NRTL at residues 131-134 of SEQ ID NO: 2, NQTN at residues 212-215 ofSEQ ID NO: 2 and/or NCSV at residues 394-397 of SEQ ID NO: 2. In anotherembodiment, proteins of the invention comprise one or more of theregions of 84P2A9 that exhibit homology to LUCA 15 and/or KIAA1152. Inanother embodiment, proteins of the invention comprise one or more ofthe 84P2A9 cAMP and cGMP-dependent protein kinase phosphorylation sitessuch as KRRS at residues 48-51 of SEQ ID NO: 2 and/or RRPS at residues102-105 of SEQ ID NO: 2. In another embodiment, proteins of theinvention comprise one or more of the 84P2A9 Protein Kinase Cphosphorylation sites such as TLR at residues 133-135 of SEQ ID NO: 2,SNK at residues 152-154 of SEQ ID NO: 2, SDR at residues 171-173 of SEQID NO: 2, TNK at residues 214-216 of SEQ ID NO: 2, SRR at residues313-315 of SEQ ID NO: 2, SSK at residues 328-330 of SEQ ID NO: 2 and/orSVR at residues 396-398 of SEQ ID NO: 2. In another embodiment, proteinsof the invention comprise one or more of the 84P2A9 casein kinase IIphosphorylation sites such as SALE at residues 10-13 of SEQ ID NO: 2,SSLE at residues 70-73 of SEQ ID NO: 2, SLEE at residues 71-74 of SEQ IDNO: 2, SDSD at residues 91-94 of SEQ ID NO: 2, TNKD at residues214-217—of SEQ ID NO: 2, SESD at residues 232-235 of SEQ ID NO: 2, SSTDat residues 240-243 of SEQ ID NO: 2, TNDE at residues 248-251 of SEQ IDNO: 2, TELD at residues 287-290 of SEQ ID NO: 2 and/or TEHD at residues374-377 of SEQ ID NO: 2. In another embodiment, proteins of theinvention comprise one or more of the N-myristoylation sites such asGSDSSL at residues 67-72 of SEQ ID NO: 2, GLFIND at residues 245-250 ofSEQ ID NO: 2, GGACGI at residues 269-274 of SEQ ID NO: 2, GGTPTS atresidues 336-341 of SEQ ID NO: 2, GTPTSM at residues 337-342 of SEQ IDNO: 2, GSLCTG at residues 409-414 of SEQ ID NO: 2, GSGLGR at residues459-464 of SEQ ID NO: 2 and/or GLGLGF at residues 481-486 of SEQ ID NO:2. In another embodiment, proteins of the invention comprise one or moreamidation sites such as RGRK at residues 45-48 of SEQ ID NO: 2 and/orRGKR at residues 113-116 of SEQ ID NO: 2. An illustrative embodiment ofsuch a polypeptide includes two or more amino acid sequences selectedfrom the group consisting of KKRK, NQTN, NCSV, TNK, SRR, SSK, SVR,GLFIND, GGACGI, GGTPTS, GTPTSM and GSLCTG (as identified above in SEQ IDNO: 2). In a preferred embodiment, the polypeptide comprises three orfour or five or six or more amino acid sequences KKRK, NQTN, NCSV, TNK,SRR, SSK, SVR, GLFTND, GGACGI, GGTPTS, GTPTSM and GSLCTG (as identifiedabove in SEQ ID NO: 2).

In another embodiment, proteins of the invention comprise one or more ofthe immunoreactive epitopes identified by a process described hereinsuch as such as those shown in Table 1. Processes for identifyingpeptides and analogues having affinities for HLA molecules and which arecorrelated as immunogenic epitopes, are well known in the art. Alsodisclosed are principles for creating analogs of such epitopes in orderto modulate immunogenicity. A variety of references are useful in theidentification of such molecules. See, for example, WO 9733602 toChestnut et al.; Sette, Immunogenetics 1999 50(3-4): 201-212; Sette etal., J. Immunol. 2001 166(2): 1389-1397; Alexander et al., Immunol. Res.18(2): 79-92; Sidney et al., Hum. Immunol. 1997 58(1): 12-20; Kondo etal., Immunogenetics 1997 45(4): 249-258; Sidney et al., J. Immunol. 1996157(8): 3480-90; and Falk et al., Nature 351: 290-6 (1991); Hunt et al.,Science 255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7 (1992);Parker et al., J. Immunol. 152:163-75 (1994)); Kast et al., 1994 152(8):3904-12; Borras-Cuesta et al., Hum. Immunol. 2000 61(3): 266-278;Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexanderet al., PMID: 7895164, UI: 95202582; O'Sullivan et al., J. Immunol. 1991147(8): 2663-2669; Alexander et al., Immunity 1994 1(9): 751-761 andAlexander et al., Immunol. Res. 1998 18(2): 79-92. The disclosures ofthese publications are hereby incorporated by reference herein in theirentireties.

Related embodiments of the invention comprise polypeptides containingcombinations of the different motifs discussed herein, where certainembodiments contain no insertions, deletions or substitutions eitherwithin the motifs or the intervening sequences of these polypeptides. Inaddition, embodiments which include a number of either N-terminal and/orC-terminal amino acid residues on either side of these motifs may bedesirable (to, for example, include a greater portion of the polypeptidearchitecture in which the motif is located). Typically the number ofN-terminal and/or C-terminal amino acid residues on either side of amotif is between about 1 to about 100 amino acid residues, preferably 5to about 50 amino acid residues.

In another embodiment of the invention, proteins of the inventioncomprise amino acid sequences that are unique to one or more 84P2A9alternative splicing variants, such as the splice variant encoded by the4.5 KB transcript that is overexpressed in prostate cancers and shown inFIG. 4. The monitoring of alternative splice variants of 84P2A9 isuseful because changes in the alternative splicing of proteins issuggested as one of the steps in a series of events that lead to theprogression of cancers (see, e.g., Carstens et al., Oncogene 15(250:3059-3065 (1997)). Consequently, monitoring of alternative splicevariants of 84P2A9 provides an additional means to evaluate syndromesassociated with perturbations in 84P2A9 gene products such as cancers.

Polypeptides comprising one or more of the 84P2A9 motifs discussedherein are useful in elucidating the specific characteristics of amalignant phenotype in view of the observation that the 84P2A9 motifsdiscussed herein are associated with growth disregulation and because84P2A9 is overexpressed in cancers (FIG. 4). Thus, the presence in aprotein of motifs related to these enzymes or molecules is relevant. Forexample, Casein kinase II, cAMP and cCMP-dependent protein kinase andProtein Kinase C for example are enzymes known to be associated with thedevelopment of the malignant phenotype (see, e.g., Chen et al., LabInvest., 78(2): 165-174 (1998); Gaiddon et al., Endocrinology 136(10):4331-4338 (1995); Hall et al., Nucleic Acids Research 24(6): 1119-1126(1996); Peterziel et al., Oncogene 18(46): 6322-6329 (1999) and O'Brian,Oncol. Rep. 5(2): 305-309 (1998)). Moreover, both glycosylation andmyristylation are protein modifications also associated with cancer andcancer progression (see, e.g., Dennis et al., Biochim. Biophys. Acta1473(1):21-34 (1999); Raju et al., Exp. Cell Res. 235(1): 145-154(1997)). Amidation is another protein modification associated withcancer and cancer progression (see, e.g., Treston et al., J. Natl.Cancer Inst. Monogr. (13): 169-175 (1992)). In addition, nuclearlocalization sequences are believed to influence the malignant potentialof a cell (see, e.g., Mirski et al., Cancer Res. 55(10): 2129-2134(1995)).

The proteins of the invention have a number of different specific uses.As 84P2A9 is shown to be highly expressed in prostate cancers (FIG. 4),these peptides/proteins are used in methods assessing the status of84P2A9 gene products in normal versus cancerous tissues and elucidatingthe malignant phenotype. Typically, polypeptides encoding specificregions of the 84P2A9 protein are used to assess the presence ofperturbations (such as deletions, insertions, point mutations etc.) inspecific regions (such regions containing a nuclear localization signal)of the 84P2A9 gene products. Exemplary assays utilize antibodies or Tcells targeting 84P2A9-related proteins comprising the amino acidresidues of one or more of the biological motifs contained within the84P2A9 polypeptide sequence in order to evaluate the characteristics ofthis region in normal versus cancerous tissues. Alternatively, 84P2A9polypeptides containing the amino acid residues of one or more of thebiological motifs contained within the 84P2A9 proteins are used toscreen for factors that interact with that region of 84P2A9.

As discussed herein, redundancy in the genetic code permits variation in84P2A9 gene sequences. In particular, one skilled in the art willrecognize specific codon preferences by a specific host species, and canadapt the disclosed sequence as preferred for a desired host. Forexample, preferred analog codon sequences typically have rare codons(i.e., codons having a useage frequency of less than about 20% in knownsequences of the desired host) replaced with higher frequency codons.Codon preferences for a specific species are calculated, for example, byutilizing codon usage tables available on the INTERNET. Nucleotidesequences that have been optimized for a particular host species byreplacing any codons having a useage frequency of less than about 20%are referred to herein as “codon optimized sequences.”

Additional sequence modifications are known to enhance proteinexpression in a cellular host. These include elimination of sequencesencoding spurious polyadenylation signals, exon/intron splice sitesignals, transposon-like repeats, and/or other such well-characterizedsequences that are deleterious to gene expression. The GC content of thesequence is adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Wherepossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures. Other useful modifications include the addition of atranslational initiation consensus sequence at the start of the openreading frame, as described in Kozak, Mol. Cell Biol., 9:5073-5080(1989). Skilled artisans understand that the general rule thateukaryotic ribosomes initiate translation exclusively at the 5′ proximalAUG codon is abrogated only under rare conditions (see, e.g., Kozak PNAS92(7): 2662-2666, (1995) and Kozak NAR 15(20): 8125-8148 (1987)).Nucleotide sequences that have been optimized for expression in a givenhost species by elimination of spurious polyadenylation sequences,elimination of exon/intron splicing signals, elimination oftransposon-like repeats and/or optimization of GC content in addition tocodon optimization are referred to herein as an “expression enhancedsequence.”

84P2A9 proteins are embodied in many forms, preferably in isolated form.A purified 84P2A9 protein molecule will be substantially free of otherproteins or molecules that impair the binding of 84P2A9 to antibody orother ligand. The nature and degree of isolation and purification willdepend on the intended use. Embodiments of an 84P2A9 protein include apurified 84P2A9 protein and a functional, soluble 84P2A9 protein. In oneembodiment, a functional soluble 84P2A9 protein or fragment thereofretains the ability to be bound by antibody, T cell or other ligand.

The invention also provides 84P2A9 proteins comprising biologicallyactive fragments of the 84P2A9 amino acid sequence corresponding to partof the 84P2A9 amino acid sequence shown in FIG. 2. Such proteins of theinvention exhibit properties of the 84P2A9 protein, such as the abilityto elicit the generation of antibodies that specifically bind an epitopeassociated with the 84P2A9 protein; to be bound by such antibodies; toelicit the activation of HTL or CTL; and/or, to be recognized by HTL orCTL.

84P2A9-related proteins are generated using standard peptide synthesistechnology or using chemical cleavage methods well known in the art.Alternatively, recombinant methods can be used to generate nucleic acidmolecules that encode an 84P2A9-related protein. In one embodiment, the84P2A9-encoding nucleic acid molecules described herein provide meansfor generating defined fragments of 84P2A9 proteins. 84P2A9 proteinfragments/subsequences are particularly useful in generating andcharacterizing domain specific antibodies (e.g., antibodies recognizingan extracellular or intracellular epitope of an 84P2A9 protein), inidentifying agents or cellular factors that bind to 84P2A9 or aparticular structural domain thereof, and in various therapeuticcontexts, including but not limited to cancer vaccines or methods ofpreparing such vaccines.

84P2A9 polypeptides containing particularly interesting structures canbe predicted and/or identified using various analytical techniques wellknown in the art, including, for example, the methods of Chou-Fasman,Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz orJameson-Wolf analysis, or on the basis of immunogenicity. Fragmentscontaining such structures are particularly useful in generating subunitspecific anti-84P2A9 antibodies, or T cells or in identifying cellularfactors that bind to 84P2A9.

Illustrating this, the binding of peptides from 84P2A9 proteins to thehuman MHC class I molecule HLA-A2 were predicted. Specifically, thecomplete amino acid sequence of the 84P2A9 protein was entered into theHLA Peptide Motif Search algorithm found in the Bioinformatics andMolecular Analysis Section (BIMAS) Web site. The HLA Peptide MotifSearch algorithm was developed by Dr. Ken Parker based on binding ofspecific peptide sequences in the groove of HLA Class I molecules andspecifically HLA-A2 (see, e.g., Falk et al. Nature 351: 290-6 (1991);Hunt et al., Science 255:1261-3 (1992); Parker et al., J. Immunol.149:3580-7 (1992); Parker et al., J. Immunol. 152:163-75 (1994)). Thisalgorithm allows location and ranking of 8-mer, 9-mer, and 10-merpeptides from a complete protein sequence for predicted binding toHLA-A2 as well as numerous other HLA Class I molecules. Many HLA class Ibinding peptides are 8-, 9-, 10 or 11-mers. For example, for class IHLA-A2, the epitopes preferably contain a leucine (L) or methionine (M)at position 2 and a valine (V) or leucine (L) at the C-terminus (see,e.g., Parker et al., J. Immunol. 149:3580-7 (1992)). Selected results of84P2A9 predicted binding peptides are shown in Table 1 below. It is tobe appreciated that every epitope predicted by the DIMAS site, orspecified by the HLA class I or class I motifs available in the art areto be applied (e.g., visually or by computer based methods, orappreciated by those of skill in the relevant art) or which become partof the art are within the scope of the invention. In Table 1, the top 10ranking candidates for each family member are shown along with theirlocation, the amino acid sequence of each specific peptide, and anestimated binding score. The binding score corresponds to the estimatedhalf-time of dissociation of complexes containing the peptide at 37° C.at pH 6.5. Peptides with the highest binding score (i.e. 63.04 for84P2A9) are predicted to be the most tightly bound to HLA Class I on thecell surface for the greatest period of time and thus represent the bestimmunogenic targets for T-cell recognition. Actual binding of peptidesto an HLA allele can be evaluated by stabilization of HLA expression onthe antigen-processing defective cell line T2 (see, e.g., Xue et al.,Prostate 30:73-8 (1997) and Peshwa et al., Prostate 36:129-38 (1998)).Immunogenicity of specific peptides can be evaluated in vitro bystimulation of CD8+ cytotoxic T lymphocytes (CTL) in the presence ofantigen presenting cells such as dendritic cells.

In an embodiment described in the examples that follow, 84P2A9 can beconveniently expressed in cells (such as 293T cells) transfected with acommercially available expression vector such as a CMV-driven expressionvector encoding 84P2A9 with a C-terminal 6×His and MYC tag(pcDNA3.1/mycHIS, Invitrogen or Tag5, GenHunter Corporation, NashvilleTerm.). The Tag5 vector provides an IgGK secretion signal that can beused to facilitate the production of a secreted 84P2A9 protein intransfected cells. The secreted HIS-tagged 84P2A9 in the culture mediacan be purified, e.g., using a nickel column using standard techniques.

Modifications of 84P2A9-related proteins such as covalent modificationsare included within the scope of this invention. One type of covalentmodification includes reacting targeted amino acid residues of an 84P2A9polypeptide with an organic derivatizing agent that is capable ofreacting with selected side chains or the N- or C-terminal residues ofthe 84P2A9. Another type of covalent modification of the 84P2A9polypeptide included within the scope of this invention comprisesaltering the native glycosylation pattern of a protein of the invention.“Altering the native glycosylation pattern” is intended for purposesherein to mean deleting one or more carbohydrate moieties found innative sequence 84P2A9 (either by removing the underlying glycosylationsite or by deleting the glycosylation by chemical and/or enzymaticmeans), and/or adding one or more glycosylation sites that are notpresent in the native sequence 84P2A9. In addition, the phrase includesqualitative changes in the glycosylation of the native proteins,involving a change in the nature and proportions of the variouscarbohydrate moieties present. Another type of covalent modification of84P2A9 comprises lining the 84P2A9 polypeptide to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol (PEG),polypropylene glycol, or polyoxyalkylenes, in the manner set forth inU.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or4,179,337.

The 84P2A9 of the present invention can also be modified in a way toform a chimeric molecule comprising 84P2A9 fused to another,heterologous polypeptide or amino acid sequence. Such a chimericmolecule can be synthesized chemically or recombinantly. A chimericmolecule can have a protein of the invention fused to anothertumor-associated antigen or fragment thereof, or can comprise fusion offragments of the 84P2A9 sequence (amino or nucleic acid) such that amolecule is created that is not, through its length, directly homologousto the amino or nucleic acid sequences respectively of FIG. 2 (SEQ IDNO: 2); such a chimeric molecule can comprise multiples of the samesubsequence of 84P2A9. A chimeric molecule can comprise a fusion of an84P2A9-related protein with a polyhistidine epitope tag, which providesan epitope to which immobilized nickel can selectively bind. The epitopetag is generally placed at the amino- or carboxyl-terminus of the84P2A9. In an alternative embodiment, the chimeric molecule can comprisea fusion of an 84P2A9-related protein with an immunoglobulin or aparticular region of an immunoglobulin. For a bivalent form of thechimeric molecule (also referred to as an “immunoadhesin”), such afusion could be to the Fc region of an IgG molecule. The Ig fusionspreferably include the substitution of a soluble (transmembrane domaindeleted or inactivated) form of an 84P2A9 polypeptide in place of atleast one variable region within an Ig molecule. In a particularlypreferred embodiment, the immunoglobulin fusion includes the hinge, CH2and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgGI molecule. Forthe production of immunoglobulin fusions see also U.S. Pat. No.5,428,130 issued Jun. 27, 1995.

84P2A9 Antibodies

Another aspect of the invention provides antibodies that bind to84P2A9-related proteins and polypeptides. Preferred antibodiesspecifically bind to an 84P2A9-related protein and will not bind (orwill bind weakly) to non-84P2A9 proteins. In another embodiment,antibodies bind 84P2A9-related proteins as well as the homologs thereof.

84P2A9 antibodies of the invention are particularly useful in prostatecancer diagnostic and prognostic assays, and imaging methodologies.Similarly, such antibodies are useful in the treatment, diagnosis,and/or prognosis of other cancers, to the extent 84P2A9 is alsoexpressed or overexpressed in other types of cancer. Moreover,intracellularly expressed antibodies (e.g., single chain antibodies) aretherapeutically useful in treating cancers in which the expression of84P2A9 is involved, such as for example advanced and metastatic prostatecancers.

The invention also provides various immunological assays useful for thedetection and quantification of 84P2A9 and mutant 84P2A9-relatedproteins. Such assays can comprise one or more 84P2A9 antibodies capableof recognizing and binding an 84P2A9 or mutant 84P2A9 protein, asappropriate, and are performed within various immunological assayformats well known in the art, including but not limited to varioustypes of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA),enzyme-linked immunofluorescent assays (ELIFA), and the like.

Related immunological but non-antibody assays of the invention alsocomprise T cell immunogenicity assays (inhibitory or stimulatory) aswell as major histocompatibility complex (MHC) binding assays. Inaddition, immunological imaging methods capable of detecting prostatecancer and other cancers expressing 84P2A9 are also provided by theinvention, including but limited to radioscintigraphic imaging methodsusing labeled 84P2A9 antibodies. Such assays are clinically useful inthe detection, monitoring, and prognosis of 84P2A9 expressing cancerssuch as prostate cancer.

84P2A9 antibodies can also be used in methods for purifying 84P2A9 andmutant 84P2A9 proteins and polypeptides and for isolating 84P2A9homologues and related molecules. For example, in one embodiment, themethod of purifying an 84P2A9 protein comprises incubating an 84P2A9antibody, which has been coupled to a solid matrix, with a lysate orother solution containing 84P2A9 under conditions that permit the 84P2A9antibody to bind to 84P2A9; washing the solid matrix to eliminateimpurities; and eluting the 84P2A9 from the coupled antibody. Other usesof the 84P2A9 antibodies of the invention include generatinganti-idiotypic antibodies that mimic the 84P2A9 protein.

Various methods for the preparation of antibodies are well known in theart. For example, antibodies can be prepared by immunizing a suitablemammalian host using an 84P2A9-related protein, peptide, or fragment, inisolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSHPress, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold SpringHarbor Press, NY (1989)). In addition, fusion proteins of 84P2A9 canalso be used, such as an 84P2A9 GST-fusion protein. In a particularembodiment, a GST fusion protein comprising all or most of the openreading frame amino acid sequence of FIG. 2 is produced and used as animmunogen to generate appropriate antibodies. In another embodiment, an84P2A9 peptide is synthesized and used as an immunogen.

In addition, naked DNA immunization techniques known in the art are used(with or without purified 84P2A9 protein or 84P2A9 expressing cells) togenerate an immune response to the encoded immunogen (for review, seeDonnelly et al., 1997, Ann. Rev. Immunol. 15: 617-648).

The amino acid sequence of 84P2-9 as shown in FIG. 2 can be used toselect specific regions of the 84P2A9 protein for generating antibodies.For example, hydrophobicity and hydrophilicity analyses of the 84P2A9amino acid sequence are used to identify hydrophilic regions in the84P2A9 structure. Regions of the 84P2A9 protein that show immunogenicstructure, as well as other regions and domains, can readily beidentified using various other methods known in the art, such asChou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultzor Jameson-Wolf analysis. Thus, each region identified by any of theseprograms/methods is within the scope of the present invention. Methodsfor the generation of 84P2A9 antibodies are further illustrated by wayof the examples provided herein.

Methods for preparing a protein or polypeptide for use as an immunogenand for preparing immunogenic conjugates of a protein with a carriersuch as BSA, KLH, or other carrier proteins are well known in the art.In some circumstances, direct conjugation using, for example,carbodiimide reagents are used; in other instances linking reagents suchas those supplied by Pierce Chemical Co., Rockford, Ill., are effective.Administration of an 84P2A9 immunogen is conducted generally byinjection over a suitable time period and with use of a suitableadjuvant, as is generally understood in the art. During the immunizationschedule, titers of antibodies can be taken to determine adequacy ofantibody formation.

84P2A9 monoclonal antibodies can be produced by various means well knownin the art. For example, immortalized cell lines that secrete a desiredmonoclonal antibody are prepared using the standard hybridoma technologyof Kohler and Milstein or modifications that immortalize producing Bcells, as is generally known. The immortalized cell lines that secretethe desired antibodies are screened by immunoassay in which the antigenis an 84P2A9-related protein. When the appropriate immortalized cellculture secreting the desired antibody is identified, the cells can beexpanded and antibodies produced either from in vitro cultures or fromascites fluid.

The antibodies or fragments can also be produced, using currenttechnology, by recombinant means. Regions that bind specifically to thedesired regions of the 84P2A9 protein can also be produced in thecontext of chimeric or complementarity determining region (CDR) graftedantibodies of multiple species origin. Humanized or human 84P2A9antibodies can also be produced and are preferred for use in therapeuticcontexts. Methods for humanizing murine and other non-human antibodies,by substituting one or more of the non-human antibody CDRs forcorresponding human antibody sequences, are well known (see for example,Jones et al., 1986, Nature 321: 522-525; Riechmnan et al., 1988, Nature332: 323-327; Verhoeyen et al., 1988, Science 239: 1534-1536). See also,Carter et al., 1993, Proc. Natl. Acad. Sci. USA 89: 4285 and Sims etal., 1993, J. Immunol. 151: 2296. Methods for producing fully humanmonoclonal antibodies include phage display and transgenic methods (forreview, see Vaughan et al., 1998, Nature Biotechnology 16: 535-539).

Fully human 84P2A9 monoclonal antibodies can be generated using cloningtechnologies employing large human Ig gene combinatorial libraries(i.e., phage display) (Griffiths and Hoogenboom, Building an in vitroimmune system: human antibodies from phage display libraries. In:Protein Engineering of Antibody Molecules for Prophylactic andTherapeutic Applications in Man. Clark, M. (Ed.), Nottingham Academic,pp 45-64 (1993); Burton and Barbas, Human Antibodies from combinatoriallibraries. Id., pp 65-82). Fully human 84P2A9 monoclonal antibodies canalso be produced using transgenic mice engineered to contain humanimmunoglobulin gene loci as described in PCT Patent ApplicationWO98/24893, Kucherlapati and Jakobovits et al., published Dec. 3, 1997(see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7(4): 607-614).This method avoids the in vitro manipulation required with phage displaytechnology and efficiently produces high affinity authentic humanantibodies.

Reactivity of 84P2A9 antibodies with an 84P2A9-related protein can beestablished by a number of well known means, including Western blot,immunoprecipitation, ELISA, and FACS analyses using, as appropriate,84P2A9-related proteins, peptides, 84P2A9-expressing cells or extractsthereof.

An 84P2A9 antibody or fragment thereof of the invention is labeled witha detectable marker or conjugated to a second molecule. Suitabledetectable markers include, but are not limited to, a radioisotope, afluorescent compound, a bioluminescent compound, chemiluminescentcompound, a metal chelator or an enzyme. Further, bi-specific antibodiesspecific for two or more 84P2A9 epitopes are generated using methodsgenerally known in the art. Homodimeric antibodies can also be generatedby cross-linking techniques known in the art (e.g., Wolff et al., CancerRes. 53: 2560-2565).

84P2A9 Transgenic Animals

Nucleic acids that encode 84P2A9 or its modified forms can also be usedto generate either transgenic animals or “knock out” animals which, inturn; are useful in the development and screening of therapeuticallyuseful reagents. In accordance with established techniques, cDNAencoding 84P2A9 can be used to clone genomic DNA encoding 84P2A9 and thegenomic sequences used to generate transgenic animals that contain cellsthat express DNA encoding 84P2A9. Methods for generating transgenicanimals, particularly animals such as mice or rats, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009. Typically, particular cells would betargeted for 84P2A9 transgene incorporation with tissue-specificenhancers.

Transgenic animals that include a copy of a transgene encoding 84P2A9can be used to examine the effect of increased expression of DNAencoding 84P2A9. Such animals can be used as tester animals for reagentsthought to confer protection from, for example, pathological conditionsassociated with its overexpression. In accordance with this facet of theinvention, an animal is treated with a reagent and a reduced incidenceof the pathological condition, compared to untreated animals bearing thetransgene, would indicate a potential therapeutic intervention for thepathological condition.

Alternatively, non-human homologues of 84P2A9 can be used to constructan 84P2A9 “knock out” animal that has a defective or altered geneencoding 84P2A9 as a result of homologous recombination between theendogenous gene encoding 84P2A9 and altered genomic DNA encoding 84P2A9introduced into an embryonic cell of the animal. For example, cDNAencoding 84P2A9 can be used to clone genomic DNA encoding 84P2A9 inaccordance with established techniques. A portion of the genomic DNAencoding 84P2A9 can be deleted or replaced with another gene, such as agene encoding a selectable marker that can be used to monitorintegration. Typically, several kilobases of unaltered flanking DNA(both at the 5′ and 3′ ends) are included in the vector [see, e.g.,Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologousrecombination vectors]. The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedDNA has homologously recombined with the endogenous DNA are selected[see, e.g., Li et al., Cell, 69:915 (1992)]. The selected cells are theninjected into a blastocyst of an animal (e.g., a mouse or rat) to formaggregation chimeras [see, e.g., Bradley, in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987), pp. 113-152]. A chimeric embryo can then be implantedinto a suitable pseudopregnant female foster animal and the embryobrought to term to create a “knock out” animal. Progeny harboring thehomologously recombined DNA in their germ cells can be identified bystandard techniques and used to breed animals in which all cells of theanimal contain the homologously recombined DNA. Knock out animals can becharacterized for instance, for their ability to defend against certainpathological conditions and for their development of pathologicalconditions due to absence of the 84P2A9 polypeptide.

Methods for the Detection of 84P2A9

Another aspect of the present invention relates to methods for detecting84P2A9 polynucleotides and 84P2A9-related proteins and variants thereof,as well as methods for identifying a cell that expresses 84P2A9. 84P2A9appears to be expressed in the LAPC xenografts that are derived fromlymph-node and bone metastasis of prostate cancer. The expressionprofile of 84P2A9 makes it a potential diagnostic marker formetastasized disease. In this context, the status of 84P2A9 geneproducts provide information useful for predicting a variety of factorsincluding susceptibility to advanced stage disease, rate of progression,and/or tumor aggressiveness. As discussed in detail below, the status of84P2A9 gene products in patient samples can be analyzed by a varietyprotocols that are well known in the art including immunohistochemicalanalysis, the variety of Northern blotting techniques including in situhybridization, RT-PCR analysis (for example on laser capturemicro-dissected samples), Western blot analysis and tissue arrayanalysis.

More particularly, the invention provides assays for the detection of84P2A9 polynucleotides in a biological sample, such as serum, bone,prostate, and other tissues, urine, semen, cell preparations, and thelike. Detectable 84P2A9 polynucleotides include, for example, an 84P2A9gene or fragments thereof, 84P2A9 mRNA, alternative splice variant84P2A9 mRNAs, and recombinant DNA or RNA molecules containing an 84P2A9polynucleotide. A number of methods for amplifying and/or detecting thepresence of 84P2A9 polynucleotides are well known in the art and can beemployed in the practice of this aspect of the invention.

In one embodiment, a method for detecting an 84P2A9 mRNA in a biologicalsample comprises producing cDNA from the sample by reverse transcriptionusing at least one primer; amplifying the cDNA so produced using an84P2A9 polynucleotides as sense and antisense primers to amplify 84P2A9cDNAs therein; and detecting the presence of the amplified 84P2A9 cDNA.Optionally, the sequence of the amplified 84P2A9 cDNA can be determined.

In another embodiment, a method of detecting an 84P2A9 gene in abiological sample comprises first isolating genomic DNA from the sample;amplifying the isolated genomic DNA using 84P2A9 polynucleotides assense and antisense primers to amplify the 84P2A9 gene therein; anddetecting the presence of the amplified 84P2A9 gene. Any number ofappropriate sense and antisense probe combinations can be designed fromthe nucleotide sequences provided for the 84P2A9 (FIG. 2) and used forthis purpose.

The invention also provides assays for detecting the presence of an84P2A9 protein in a tissue of other biological sample such as serum,bone, prostate, and other tissues, urine, cell preparations, and thelike. Methods for detecting an 84P2A9 protein are also well known andinclude, for example, immunoprecipitation, immunohistochemical analysis,Western Blot analysis, molecular binding assays, ELISA, ELIFA and thelike. For example, in one embodiment, a method of detecting the presenceof an 84P2A9 protein in a biological sample comprises first contactingthe sample with an 84P2A9 antibody, an 84P2A9-reactive fragment thereof,or a recombinant protein containing an antigen binding region of an84P2A9 antibody; and then detecting the binding of 84P2A9 protein in thesample thereto.

Methods for identifying a cell that expresses 84P2A9 are also provided.In one embodiment, an assay for identifying a cell that expresses an84P2A9 gene comprises detecting the presence of 84P2A9 mRNA in the cell.Methods for the detection of particular mRNAs in cells are well knownand include, for example, hybridization assays using complementary DNAprobes (such as in situ hybridization using labeled 84P2A9 riboprobes,Northern blot and related techniques) and various nucleic acidamplification assays (such as RT-PCR using complementary primersspecific for 84P2A9, and other amplification type detection methods,such as, for example, branched DNA, SISBA, TMA and the like).Alternatively, an assay for identifying a cell that expresses an 84P2A9gene comprises detecting the presence of 84P2A9 protein in the cell orsecreted by the cell. Various methods for the detection of proteins arewell known in the art and are employed for the detection of 84P2A9proteins and 84P2A9 expressing cells.

84P2A9 expression analysis is also useful as a tool for identifying andevaluating agents that modulate 84P2A9 gene expression. For example,84P2A9 expression is significantly upregulated in prostate cancer, andis also expressed in other cancers including prostate, testis, kidney,brain, bone, skin, ovarian, breast, pancreas, colon, lymphocytic andlung cancers. Identification of a molecule or biological agent thatcould inhibit 84P2A9 expression or over-expression in cancer cells is oftherapeutic value. For example, such an agent can be identified by usinga screen that quantifies 84P2A9 expression by RT-PCR, nucleic acidhybridization or antibody binding.

Monitoring the Status of 84P2A9 and its Products

Assays that evaluate the status of the 84P2A9 gene and 84P2A9 geneproducts in an individual can provide information on the growth oroncogenic potential of a biological sample from this individual. Forexample, because 84P2A9 mRNA is so highly expressed in prostate cancers(as well as the other cancer tissues shown for example in FIGS. 4-8) ascompared to normal prostate tissue, assays that evaluate the relativelevels of 84P2A9 mRNA transcripts or proteins in a biological sample canbe used to diagnose a disease associated with 84P2A9 disregulation suchas cancer and can provide prognostic information useful in definingappropriate therapeutic options.

Because 84P2A9 is expressed, for example, in various prostate cancerxenograft tissues and cancer cell lines, and cancer patient samples, theexpression status of 84P2A9 can provide information useful fordetermining information including the presence, stage and location ofdysplastic, precancerous and cancerous cells, predicting susceptibilityto various stages of disease, and/or for gauging tumor aggressiveness.Moreover, the expression profile makes it useful as an imaging reagentfor metastasized disease. Consequently, an important aspect of theinvention is directed to the various molecular prognostic and diagnosticmethods for examining the status of 84P2A9 in biological samples such asthose from individuals suffering from, or suspected of suffering from apathology characterized by disregulated cellular growth such as cancer.

Oncogenesis is known to be a multistep process where cellular growthbecomes progressively disregulated and cells progress from a normalphysiological state to precancerous and then cancerous states (see,e.g., Alers et al., Lab Invest. 77(5): 437-438 (1997) and Isaacs et al.,Cancer Surv. 23: 19-32 (1995)). In this context, examining a biologicalsample for evidence of disregulated cell growth (such as aberrant 84P2A9expression in prostate cancers) can allow the early detection of suchaberrant cellular physiology before a pathology such as cancer hasprogressed to a stage at which therapeutic options are more limited. Insuch examinations, the status of 84P2A9 in a biological sample ofinterest (such as one suspected of having disregulated cell growth) canbe compared, for example, to the status of 84P2A9 in a correspondingnormal sample (e.g. a sample from that individual (or alternativelyanother individual that is not effected by a pathology, for example onenot suspected of having disregulated cell growth). Alterations in thestatus of 84P2A9 in the biological sample of interest (as compared tothe normal sample) provides evidence of disregulated cellular growth. Inaddition to using a biological sample that is not effected by apathology as a normal sample, one can also use a predetermined normativevalue such as a predetermined normal level of mRNA expression (see,e.g., Grever et al., J. Comp. Neurol. 1996 Dec. 9; 376(2):306-14 andU.S. Pat. No. 5,837,501) to compare 84P2A9 in normal versus suspectsamples.

The term “status” in this context is used according to its art acceptedmeaning and refers to the condition or state of a gene and its products.Typically, skilled artisans use a number of parameters to evaluate thecondition or state of a gene and its products. These include, but arenot limited to the location of expressed gene products (including thelocation of 84P2A9 expressing cells) as well as the, level, andbiological activity of expressed gene products (such as 84P2A9 mRNApolynucleotides and polypeptides). Alterations in the status of 84P2A9can be evaluated by a wide variety of methodologies well known in theart, typically those discussed herein. Typically an alteration in thestatus of 84P2A9 comprises a change in the location of 84P2A9 and/or84P2A9 expressing cells and/or an increase in 84P2A9 mRNA and/or proteinexpression.

As discussed in detail herein, in order to identify a condition orphenomenon associated with disregulated cell growth, the status of84P2A9 in a biological simple is evaluated by a number of methodsutilized by skilled artisans including, but not limited to genomicSouthern analysis (to examine, for example perturbations in the 84P2A9gene), Northern analysis and/or PCR analysis of 84P2A9 mRNA (to examine,for example alterations in the polynucleotide sequences or expressionlevels of 84P2A9 mRNAs), and Western and/or immunohistochemical analysis(to examine, for example alterations in polypeptide sequences,alterations in polypeptide localization within a sample, alterations inexpression levels of 84P2A9 proteins and/or associations of 84P2A9proteins with polypeptide binding partners). Detectable 84P2A9polynucleotides include, for example, an 84P2A9 gene or fragmentsthereof, 84P2A9 mRNA, alternative splice variants 84P2A9 mRNAs, andrecombinant DNA or RNA molecules containing an 84P2A9 polynucleotide.

The expression profile of 84P2A9 makes it a diagnostic marker for localand/or metastasized disease. In particular, the status of 84P2A9provides information useful for predicting susceptibility to particulardisease stages, progression, and/or tumor aggressiveness. The inventionprovides methods and assays for determining 84P2A9 status and diagnosingcancers that express 84P2A9, such as cancers of the prostate, bladder,testis, ovaries, breast, pancreas, colon and lung. 84P2A9 status inpatient samples can be analyzed by a number of means well known in theart including without limitation, immunohistochemical analysis, in situhybridization, RT-PCR analysis on laser capture micro-dissected samples,Western blot analysis of clinical samples and cell lines, and tissuearray analysis. Typical protocols for evaluating the status of the84P2A9 gene and gene products can be found, for example in Ausubul etal. eds., 1995, Current Protocols In Molecular Biology, Units 2[Northern Blotting], 4 [Southern Blotting], 15 [Immunoblotting] and 18[PCR Analysis].

As described above, the status of 84P2A9 in a biological sample can beexamined by a number of well known procedures in the art. For example,the status of 84P2A9 in a biological sample taken from a specificlocation in the body can be examined by evaluating the sample for thepresence or absence of 84P2A9 expressing cells (e.g. those that express84P2A9 mRNAs or proteins). This examination can provide evidence ofdisregulated cellular growth, for example, when 84P2A9 expressing cellsare found in a biological sample that does not normally contain suchcells (such as a lymph node). Such alterations in the status of 84P2A9in a biological sample are often associated with disregulated cellulargrowth. Specifically, one indicator of disregulated cellular growth isthe metastases of cancer cells from an organ of origin (such as thetestis or prostate gland) to a different area of the body (such as alymph node). In this context, evidence of disregulated cellular growthis important for example because occult lymph node metastases can bedetected in a substantial proportion of patients with prostate cancer,and such metastases are associated with known predictors of diseaseprogression (see, e.g., Murphy et al., Prostate 42(4): 315-317 (2000);Su et al., Semin. Surg. Oncol. 18(1): 17-28 (2000) and Freeman et al., JUrol 1995 August; 154(2 Pt 1):474-8).

In one aspect, the invention provides methods for monitoring 84P2A9 geneproducts by determining the status of 84P2A9 gene products expressed bycells in a test tissue sample from an individual suspected of having adisease associated with disregulated cell growth (such as hyperplasia orcancer) and then comparing the status so determined to the status of84P2A9 gene products in a corresponding normal sample, the presence ofaberrant 84P2A9 gene products in the test sample relative to the normalsample providing an indication of the presence of disregulated cellgrowth within the cells of the individual.

In another aspect, the invention provides assays useful in determiningthe presence of cancer in an individual comprising detecting asignificant increase in 84P2A9 mRNA or protein expression in a test cellor tissue sample relative to expression levels in the correspondingnormal cell or tissue. The presence of 84P2A9 mRNA can, for example, beevaluated in tissue samples including but not limited to prostate,testis, kidney, brain, bone, skin, ovarian, breast, pancreas, colon,lymphocytic and lung tissues (see, e.g., FIG. 48). The presence ofsignificant 84P2A9 expression in any of these tissues is useful toindicate the emergence, presence and/or severity of a cancer, since thecorresponding normal tissues do not express 84P2A9 mRNA or express it atlower levels.

In a related embodiment, 84P2A9 status is determined at the proteinlevel rather than at the nucleic acid level. For example, such a methodor assay comprises determining the level of 84P2A9 protein expressed bycells in a test tissue sample and comparing the level so determined tothe level of 84P2A9 expressed in a corresponding normal sample. In oneembodiment, the presence of 84P2A9 protein is evaluated, for example,using immunohistochemical methods. 84P2A9 antibodies or binding partnerscapable of detecting 84P2A9 protein expression are used in a variety ofassay formats well known in the art for this purpose.

In other related embodiments, one can evaluate the status 84P2A9nucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules such asinsertions, deletions, substitutions and the like. Such embodiments areuseful because perturbations in the nucleotide and amino acid sequencesare observed in a large number of proteins associated with a growthdisregulated phenotype (see, e.g., Marrogi et al., 1999, J. Cutan.Pathol. 26(8):369-378). For example, a mutation is the sequence of84P2A9 may be indicative of the presence or promotion of a tumor. Suchassays can therefore have diagnostic and predictive value where amutation in 84P2A9 indicates a potential loss of function or increase intumor growth.

A wide variety of assays for observing perturbations in nucleotide andamino acid sequences are well known in the art. For example, the sizeand structure of nucleic acid or amino acid sequences of 84P2A9 geneproducts are observed by the Northern, Southern, Western, PCR and DNAsequencing protocols discussed herein. In addition, other methods forobserving perturbations in nucleotide and amino acid sequences such assingle strand conformation polymorphism analysis are well known in theart (see, e.g., U.S. Pat. Nos. 5,382,510 and 5,952,170).

In another embodiment, one can examine the methylation status of the84P2A9 gene in a biological sample. Aberrant demethylation and/orhypermethylation of CpG islands in gene 5′ regulatory regions frequentlyoccurs in immortalized and transformed cells and can result in alteredexpression of various genes. For example, promoter hypermethylation ofthe pi-class glutathione S-transferase (a protein expressed in normalprostate but not expressed in >90% of prostate carcinomas) appears topermanently silence transcription of this gene and is the mostfrequently detected genomic alteration in prostate carcinomas (De Marzoet al., Am. J. Pathol. 155(6): 1985-1992 (1999)). In addition, thisalteration is present in at least 70% of cases of high-grade prostaticintraepithelial neoplasia (PIN) (Brooks et al, Cancer Epidemiol.Biomarkers Prev., 1998, 7:531-536). In another example, expression ofthe LAGE-I tumor specific gene (which is not expressed in normalprostate but is expressed in 25-50% of prostate cancers) is induced bydeoxy-azacytidine in lymphoblastoid cells, suggesting that tumoralexpression is due to demethylation (Lethe et al., Int. J. Cancer 76(6):903-908 (1998)). In this context, a variety of assays for examiningmethylation status of a gene are well known in the art. For example, onecan utilize, in Southern hybridization approaches, methylation-sensitiverestriction enzymes which can not cleave sequences that containmethylated CpG sites, in order to assess the overall methylation statusof CpG islands. In addition, MSP (methylation specific PCR) can rapidlyprofile the methylation status of all the CpG sites present in a CpGisland of a given gene. This procedure involves initial modification ofDNA by sodium bisulfite (which will convert all unmethylated cytosinesto uracil) followed by amplification using primers specific formethylated versus unmethylated DNA. Protocols involving methylationinterference can also be found for example in Current Protocols InMolecular Biology, Units 12, Frederick M. Ausubul et al. eds., 1995.

Gene amplification provides an additional method of assessing the statusof 84P2A9, a locus that maps to 1q32.3, a region shown to be perturbedin a variety of cancers. Gene amplification is measured in a sampledirectly, for example, by conventional Southern blotting or Northernblotting to quantitate the transcription of mRNA (Thomas, 1980, Proc.Natl. Acad. Sci. USA, 77:5201-5205), dot blotting DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies are employed thatrecognize specific duplexes, including DNA duplexes, RNA duplexes, andDNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turnare labeled and the assay carried out where the duplex is bound to asurface, so that upon the formation of duplex on the surface, thepresence of antibody bound to the duplex can be detected.

In addition to the tissues discussed herein, biopsied tissue orperipheral blood can be conveniently assayed for the presence of cancercells, including but not limited to prostate, testis, kidney, brain,bone, skin, ovarian, breast, pancreas, colon, lymphocytic and lungcancers using for example, Northern, dot blot or RT-PCR analysis todetect 84P2A9 expression (see, e.g., FIGS. 4-8). The presence of RT-PCRamplifiable 84P2A9 mRNA provides an indication of the presence of thecancer. RT-PCR detection assays for tumor cells in peripheral blood arecurrently being evaluated for use in the diagnosis and management of anumber of human solid tumors. In the prostate cancer field, theseinclude RT-PCR assays for the detection of cells expressing PSA and PSM(Verkaik et al., 1997, Urol. Res. 25:373-384; Ghossein et al., 1995, J.Clin. Oncol. 13:1195-2000; Heston et al., 1995, Clin. Chem.41:1687-1688). RT-PCR assays are well known in the art.

A related aspect of the invention is directed to predictingsusceptibility to developing cancer in an individual. In one embodiment,a method for predicting susceptibility to cancer comprises detecting84P2A9 mRNA or 84P2A9 protein in a tissue sample, its presenceindicating susceptibility to cancer, wherein the degree of 84P2A9 mRNAexpression present correlates to the degree of susceptibility. In aspecific embodiment, the presence of 84P2A9 in prostate or other tissueis examined, with the presence of 84P2A9 in the sample providing anindication of prostate cancer susceptibility (or the emergence orexistence of a prostate tumor). In a closely related embodiment, one canevaluate the integrity 84P2A9 nucleotide and amino acid sequences in abiological sample in order to identify perturbations in the structure ofthese molecules such as insertions, deletions, substitutions and thelike, with the presence of one or more perturbations in 84P2A9 geneproducts in the sample providing an indication of cancer susceptibility(or the emergence or existence of a tumor).

Another related aspect of the invention is directed to methods forgauging tumor aggressiveness. In one embodiment, a method for gaugingaggressiveness of a tumor comprises determining the level of 84P2A9 mRNAor 84P2A9 protein expressed by cells in a sample of the tumor, comparingthe level so determined to the level of 84P2A9 mRNA or 84P2A9 proteinexpressed in a corresponding normal tissue taken from the sameindividual or a normal tissue reference sample, wherein the degree of84P2A9 mRNA or 84P2A9 protein expression in the tumor sample relative tothe normal sample indicates the degree of aggressiveness. In a specificembodiment, aggressiveness of a tumor is evaluated by determining theextent to which 84P2A9 is expressed in the tumor cells, with higherexpression levels indicating more aggressive tumors. In a closelyrelated embodiment, one can evaluate the integrity of 84P2A9 nucleotideand amino acid sequences in a biological sample in order to identifyperturbations in the structure of these molecules such as insertions,deletions, substitutions and the like, with the presence of one or moreperturbations indicating more aggressive tumors.

Yet another related aspect of the invention is directed to methods forobserving the progression of a malignancy in an individual over time. Inone embodiment, methods for observing the progression of a malignancy inan individual over time comprise determining the level of 84P2A9 mRNA or84P2A9 protein expressed by cells in a sample of the tumor, comparingthe level so determined to the level of 84P2A9 mRNA or 84P2A9 proteinexpressed in an equivalent tissue sample taken from the same individualat a different time, wherein the degree of 84P2A9 mRNA or 84P2A9 proteinexpression in the tumor sample over time provides information on theprogression of the cancer. In a specific embodiment, the progression ofa cancer is evaluated by determining the extent to which 84P2A9expression in the tumor cells alters over time, with higher expressionlevels indicating a progression of the cancer. Also, one can evaluatethe integrity 84P2A9 nucleotide and amino acid sequences in a biologicalsample in order to identify perturbations in the structure of thesemolecules such as insertions, deletions, substitutions and the like,with the presence of one or more perturbations indicating a progressionof the cancer.

The above diagnostic approaches can be combined with any one of a widevariety of prognostic and diagnostic protocols known in the art. Forexample, another embodiment of the invention disclosed herein isdirected to methods for observing a coincidence between the expressionof 84P2A9 gene and 84P2A9 gene products (or perturbations in 84P2A9 geneand 84P2A9 gene products) and a factor that is associated withmalignancy as a means of diagnosing and prognosticating the status of atissue sample. In this context, a wide variety of factors associatedwith malignancy can be utilized such as the expression of genesassociated with malignancy (e.g. PSA, PSCA and PSM expression forprostate cancer etc.) as well as gross cytological observations (see,e.g., Bocking et al., 1984, Anal. Quant. Cytol. 6(2):74-88; Eptsein,1995, Hum. Pathol. 26(2):223-9; Thorson et al., 1998, Mod. Pathol.11(6):543-51; Baisden et al., 1999, Am. J. Surg. Pathol. 23(8):918-24).Methods for observing a coincidence between the expression of 84P2A9gene and 84P2A9 gene products (or perturbations in 84P2A9 gene and84P2A9 gene products) and an additional factor that is associated withmalignancy are useful, for example, because the presence of a set ofspecific factors that coincide with disease provides information crucialfor diagnosing and prognosticating the status of a tissue sample.

In a typical embodiment, methods for observing a coincidence between theexpression of 84P2A9 gene and 84P2A9 gene products (or perturbations in84P2A9 gene and 84P2A9 gene products) and a factor that is associatedwith malignancy entails detecting the overexpression of 84P2A9 mRNA orprotein in a tissue sample, detecting the overexpression of PSA mRNA orprotein in a tissue sample, and observing a coincidence of 84P2A9 mRNAor protein and PSA mRNA or protein overexpression. In a specificembodiment, the expression of 84P2A9 and PSA mRNA in prostate tissue isexamined. In a preferred embodiment, the coincidence of 84P2A9 and PSAmRNA overexpression in the sample provides an indication of prostatecancer, prostate cancer susceptibility or the emergence or status of aprostate tumor.

Methods for detecting and quantifying the expression of 84P2A9 mRNA orprotein are described herein and use of standard nucleic acid andprotein detection and quantification technologies is well known in theart. Standard methods for the detection and quantification of 84P2A9mRNA include in situ hybridization using labeled 84P2A9 riboprobes,Northern blot and related techniques using 84P2A9 polynucleotide probes,RT-PCR analysis using primers specific for 84P2A9, and otheramplification type detection methods, such as, for example, branchedDNA, SISBA, TMA and the like. In a specific embodiment, semiquantitative RT-PCR is used to detect and quantify 84P2A9 mRNAexpression as described in the Examples that follow. Any number ofprimers capable of amplifying 84P2A9 can be used for this purpose,including but not limited to the various primer sets specificallydescribed herein. Standard methods for the detection and quantificationof protein are used for this purpose. In a specific embodiment,polyclonal or monoclonal antibodies specifically reactive with thewild-type 84P2A9 protein can be used in an immunohistochemical assay ofbiopsied tissue.

Identifying Molecules that Interact with 84P2A9

The 84P2A9 protein sequences disclosed herein allow the skilled artisanto identify proteins, small molecules and other agents that interactwith 84P2A9 and pathways activated by 84P2A9 via any one of a variety ofart accepted protocols. For example, one can utilize one of the varietyof so-called interaction trap systems (also referred to as the“two-hybrid assay”). In such systems, molecules that interactreconstitute a transcription factor which directs expression of areporter gene, whereupon the expression of the reporter gene is assayed.Typical systems identify protein-protein interactions in vivo throughreconstitution of a eukaryotic transcriptional activator and aredisclosed for example in U.S. Pat. Nos. 5,955,280, 5,925,523, 5,846,722and 6,004,746.

Alternatively one can identify molecules that interact with 84P2A9protein sequences by screening peptide libraries. In such methods,peptides that bind to selected receptor molecules such as 84P2A9 areidentified by screening libraries that encode a random or controlledcollection of amino acids. Peptides encoded by the libraries areexpressed as fusion proteins of bacteriophage coat proteins, thebacteriophage particles are then screened against the receptors ofinterest.

Accordingly, peptides having a wide variety of uses, such as therapeuticor diagnostic reagents, can thus be identified without any priorinformation on the structure of the expected ligand or receptormolecule. Typical peptide libraries and screening methods that can beused to identify molecules that interact with 84P2A9 protein sequencesare disclosed for example in U.S. Pat. Nos. 5,723,286 and 5,733,731.

Alternatively, cell lines that express 84P2A9 are used to identifyprotein-protein interactions mediated by 84P2A9. Such interactions canbe examined using immunoprecipitation techniques as shown by others(Hamilton B J, et al. Biochem. Biophys. Res. Commun. 1999, 261:646-51).Typically 84P2A9 protein can be immunoprecipitated from 84P2A9expressing prostate cancer cell lines using anti-84P2A9 antibodies.Alternatively, antibodies against His-tag can be used in a cell lineengineered to express 84P2A9 (vectors mentioned above). Theimmunoprecipitated complex can be examined for protein association byprocedures such as Western blotting, 35-methionine labeling of proteins,protein microsequencing, silver staining and two dimensional gelelectrophoresis.

Small molecules that interact with 84P2A9 can be identified throughrelated embodiments of such screening assays. For example, smallmolecules can be identified that interfere with protein function,including molecules that interfere with 84P2A9's ability to mediatephosphorylation and de-phosphorylation, second messenger signaling andtumorigenesis. Typical methods are discussed for example in U.S. Pat.No. 5,928,868 and include methods for forming hybrid ligands in which atleast one ligand is a small molecule. In an illustrative embodiment, thehybrid ligand is introduced into cells that in turn contain a first anda second expression vector. Each expression vector includes DNA forexpressing a hybrid protein that encodes a target protein linked to acoding sequence for a transcriptional module. The cells further containa reporter gene, the expression of which is conditioned on the proximityof the first and second hybrid proteins to each other, an event thatoccurs only if the hybrid ligand binds to target sites on both hybridproteins. Those cells that express the reporter gene are selected andthe unknown small molecule or the unknown hybrid protein is identified.

A typical embodiment of this invention consists of a method of screeningfor a molecule that interacts with an 84P2A9 amino acid sequence shownin FIG. 1 (SEQ ID NO: 2), comprising the steps of contacting apopulation of molecules with the 84P2A9 amino acid sequence, allowingthe population of molecules and the 84P2A9 amino acid sequence tointeract under conditions that facilitate an interaction, determiningthe presence of a molecule that interacts with the 84P2A9 amino acidsequence and then separating molecules that do not interact with the84P2A9 amino acid sequence from molecules that do interact with the84P2A9 amino acid sequence. In a specific embodiment, the method furtherincludes purifying a molecule that interacts with the 84P2A9 amino acidsequence. In a preferred embodiment, the 84P2A9 amino acid sequence iscontacted with a library of peptides.

Therapeutic Methods and Compositions

The identification of 84P2A9 as a protein that is normally prostate andtestis-related and which is also expressed in cancers of the prostate(and other cancers), opens a number of therapeutic approaches to thetreatment of such cancers. As discussed herein, it is possible that84P2A9 functions as a transcription factor involved in activatingtumor-promoting genes or repressing genes that block tumorigenesis.

The expression profile of 84P2A9 is reminiscent of the Cancer-Testis(CT) antigens or MAGE antigens, which are testis-related genes that areup-regulated in melanomas and other cancers (Van den Eynde and Boon, IntJ Clin Lab Res. 27:81-86, 1997). Due to their tissue-specific expressionand high expression levels in cancer, the MAGE antigens are currentlybeing investigated as targets for cancer vaccines (Durrant, AnticancerDrugs 8:727-733, 1997; Reynolds et al., Int J Cancer 72:972-976, 1997).The expression pattern of 84P2A9 provides evidence that it is likewisean ideal target for a cancer vaccine approach to prostate cancer. Itsstructural features indicate that it may be a transcription factor, andprovide evidence that 84P2A9 is a small molecule target

Accordingly, therapeutic approaches aimed at inhibiting the activity ofthe 84P2A9 protein are expected to be useful for patients suffering fromprostate cancer, testicular cancer, and other cancers expressing 84P2A9.These therapeutic approaches generally fall into two classes. One classcomprises various methods for inhibiting the binding or association ofthe 84P2A9 protein with its binding partner or with others proteins.Another class comprises a variety of methods for inhibiting thetranscription of the 84P2A9 gene or translation of 84P2A9 mRNA.

84P2A9 as a Target for Antibody-Based Therapy

The structural features of 84P2A9 indicate that this molecule is anattractive target for antibody-based therapeutic strategies. A number oftypical antibody strategies are known in the art for targeting bothextracellular and intracellular molecules (see, e.g., complement andADCC mediated killing as well as the use of intrabodies discussedherein). Because 84P2A9 is expressed by cancer cells of various lineagesand not by corresponding normal cells, systemic administration of84P2A9-immunoreactive compositions would be expected to exhibitexcellent sensitivity without toxic, non-specific and/or non-targeteffects caused by binding of the immunotherapeutic molecule tonon-target organs and tissues. Antibodies specifically reactive withdomains of 84P2A9 can be useful to treat 84P2A9-expressing cancerssystemically, either as conjugates with a toxin or therapeutic agent, oras naked antibodies capable of inhibiting cell proliferation orfunction.

84P2A9 antibodies can be introduced into a patient such that theantibody binds to 84P2A9 and modulates or perturbs a function such as aninteraction with a binding partner and consequently mediates growthinhibition and/or destruction of the tumor cells and/or inhibits thegrowth of the tumor cells. Mechanisms by which such antibodies exert atherapeutic effect can include complement-mediated cytolysis,antibody-dependent cellular cytotoxicity, modulating the physiologicalfunction of 84P2A9, inhibiting ligand binding or signal transductionpathways, modulating tumor cell differentiation, altering tumorangiogenesis factor profiles, and/or by inducing apoptosis.

Those skilled in the art understand that antibodies can be used tospecifically target and bind immunogenic molecules such as animmunogenic region of the 84P2A9 sequence shown in FIG. 1. In addition,skilled artisans understand that it is routine to conjugate antibodiesto cytotoxic agents. In this context, skilled artisans understand thatwhen cytotoxic and/or therapeutic agents are delivered directly to cellsby conjugating them to antibodies specific for a molecule expressed bythat cell (e.g. 84P2A9), it is reasonable to expect that the cytotoxicagent will exert its known biological effect (e.g. cytotoxicity) onthose cells.

A wide variety of compositions and methods for using antibodiesconjugated to cytotoxic agents to kill cells are known in the art. Inthe context of cancers, typical methods entail administering to ananimal having a tumor a biologically effective amount of a conjugatecomprising a selected cytotoxic and/or therapeutic agent linked to atargeting agent (e.g. an anti-84P2A9 antibody) that binds to a marker(e.g. 84P2A9) expressed, accessible to binding or localized on the cellsurfaces. A typical embodiment consists of a method of delivering acytotoxic and/or therapeutic agent to a cell expressing 84P2A9comprising conjugating the cytotoxic agent to an antibody thatimmunospecifically binds to an 84P2A9 epitope and exposing the cell tothe antibody-agent conjugate. Another specific illustrative embodimentconsists of a method of treating an individual suspected of sufferingfrom metastasized cancer comprising the step of administeringparenterally to said individual a pharmaceutical composition comprisinga therapeutically effective amount of an antibody conjugated to acytotoxic and/or therapeutic agent.

Cancer immunotherapy using anti-84P2A9 antibodies may follow theteachings generated from various approaches that have been successfullyemployed in the treatment of other types of cancer, including but notlimited to colon cancer (Adlen et al., 1998, Crit. Rev. Immunol.18:133-138), multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186;Tsunenari et al., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk etal., 1992, Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al.,1996, J. Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhonget al., 1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al.,1994, Cancer Res. 54:6160-6166; Velders et al., 1995, Cancer Res.55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clin.Immunol. 11:117-127). Some therapeutic approaches involve conjugation ofnaked antibody to a toxin, such as the conjugation of ¹³¹I to anti-CD20antibodies (e.g., Rituxan™, IDEC Pharmaceuticals Corp.), while othersinvolve co-administration of antibodies and other therapeutic agents,such as Herceptin™ (trastuzumab) with paclitaxel (Genentech, Inc.). Fortreatment of prostate cancer, for example, 84P2A9 antibodies can beadministered in conjunction with radiation, chemotherapy or hormoneablation.

Although 84P2A9 antibody therapy is useful for all stages of cancer,antibody therapy is particularly appropriate in advanced or metastaticcancers. Treatment with the antibody therapy of the invention isindicated for patients who have received one or more rounds ofchemotherapy, while combining the antibody therapy of the invention witha chemotherapeutic or radiation regimen is preferred for patients whohave not received chemotherapeutic treatment Additionally, antibodytherapy can enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well.

It is desirable for some cancer patients to be evaluated for thepresence and level of 84P2A9 expression, preferably usingimmunohistochemical assessments of tumor tissue, quantitative 84P2A9imaging, or other techniques capable of reliably indicating the presenceand degree of 84P2A9 expression. Immunohistochemical analysis of tumorbiopsies or surgical specimens is preferred for this purpose. Methodsfor immunohistochemical analysis of tumor tissues are well known in theart Anti-84P2A9 monoclonal antibodies useful in treating prostate andother cancers include those that are capable of initiating a potentimmune response against the tumor or those that are directly cytotoxic.In this regard, anti-84P2A9 monoclonal antibodies (mAbs) can elicittumor cell lysis by either complement-mediated or antibody-dependentcell cytotoxicity (ADCC) mechanisms, both of which require an intact Fcportion of the immunoglobulin molecule for interaction with effectorcell Fc receptor sites or complement proteins. In addition, anti-84P2A9mAbs that exert a direct biological effect on tumor growth are useful inthe practice of the invention. Potential mechanisms by which suchdirectly cytotoxic mAbs can act include inhibition of cell growth,modulation of cellular differentiation, modulation of tumor angiogenesisfactor profiles, and the induction of apoptosis. The mechanism by whicha particular anti-84P2A9 mAb exerts an anti-tumor effect is evaluatedusing any number of in vitro assays designed to determine cell deathsuch as ADCC, ADMMC, complement-mediated cell lysis, and so forth, as isgenerally known in the art.

The use of murine or other non-human monoclonal antibodies, orhuman/mouse chimeric mAbs can induce moderate to strong immune responsesin some patients. In some cases, this will result in clearance of theantibody from circulation and reduced efficacy. In the most severecases, such an immune response can lead to the extensive formation ofimmune complexes which, potentially, can cause renal failure.Accordingly, preferred monoclonal antibodies used in the practice of thetherapeutic methods of the invention are those that are either fullyhuman or humanized and that bind specifically to the target 84P2A9antigen with high affinity but exhibit low or no antigenicity in thepatient.

Therapeutic methods of the invention contemplate the administration ofsingle anti-84P2A9 mAbs as well as combinations, or cocktails, ofdifferent mAbs. Such mAb cocktails can have certain advantages inasmuchas they contain mAbs that target different epitopes, exploit differenteffector mechanisms or combine directly cytotoxic mAbs with mAbs thatrely on immune effector functionality. Such mAbs in combination canexhibit synergistic therapeutic effects. In addition, the administrationof anti-84P2A9 mAbs can be combined with other therapeutic agents,including but not limited to various chemotherapeutic agents,androgen-blockers, and immune modulators (e.g., IL-2, GM-CSF). Theanti-84P2A9 mAbs are administered in their “naked” or unconjugated form,or can have therapeutic agents conjugated to them.

The anti-84P2A9 antibody formulations are administered via any routecapable of delivering the antibodies to the tumor site. Potentiallyeffective routes of administration include, but are not limited to,intravenous, intraperitoneal, intramuscular, intratumor, intradermal,and the like. Treatment will generally involve the repeatedadministration of the anti-84P2A9 antibody preparation via an acceptableroute of administration such as intravenous injection (IV), typically ata dose in the range of about 0.1 to about 10 mg/kg body weight. Doses inthe range of 10-500 mg mAb per week are effective and well tolerated.

Based on clinical experience with the Herceptin mAb in the treatment ofmetastatic breast cancer, an initial loading dose of approximately 4mg/kg patient body weight IV followed by weekly doses of about 2 mg/kgIV of the anti-84P2A9 mAb preparation represents an acceptable dosingregimen. Preferably, the initial loading dose is administered as a 90minute or longer infusion. The periodic maintenance dose is administeredas a 30 minute or longer infusion, provided the initial dose was welltolerated. However, as one of skill in the art will understand, variousfactors will influence the ideal dose regimen in a particular case. Suchfactors can include, for example, the binding affinity and half life ofthe Ab or mAbs used, the degree of 84P2A9 expression in the patient, theextent of circulating shed 84P2A9 antigen, the desired steady-stateantibody concentration level frequency of treatment, and the influenceof chemotherapeutic agents used in combination with the treatment methodof the invention, as well as the health status of a particular patient.

Optionally, patients should be evaluated for the levels of 84P2A9 in agiven sample (e.g. the levels of circulating 84P2A9 antigen and/or84P2A9 expressing cells) in order to assist in the determination of themost effective dosing regimen and related factors. Such evaluations arealso be used for monitoring purposes throughout therapy, and are usefulto gauge therapeutic success in combination with evaluating otherparameters (such as serum PSA levels in prostate cancer therapy).

Inhibition of 84P2A9 Protein Function

Within the first class of therapeutic approaches, the invention includesvarious methods and compositions for inhibiting the binding of 84P2A9 toits binding partner or its association with other protein(s) as well asmethods for inhibiting 84P2A9 function.

Inhibition of 84P2A9 with Intracellular Antibodies

In one approach, recombinant vectors encoding single chain antibodiesthat specifically bind to 84P2A9 are introduced into 84P2A9 expressingcells via gene transfer technologies, wherein the encoded single chainanti-84P2A9 antibody is expressed intracellularly, binds to 84P2A9protein, and thereby inhibits its function. Methods for engineering suchintracellular single chain antibodies are well known. Such intracellularantibodies, also known as “intrabodies”, are specifically targeted to aparticular compartment within the cell, providing control over where theinhibitory activity of the treatment will be focused. This technologyhas been successfully applied in the art (for review, see Richardson andMarasco, 1995, TIBTECH vol. 13). Intrabodies have been shown tovirtually eliminate the expression of otherwise abundant cell surfacereceptors. See, for example, Richardson et al., 1995, Proc. Natl. Acad.Sci. USA 92: 3137-3141; Beerli et al., 1994, J. Biol. Chem. 289:23931-23936; Deshane et al., 1994, Gene Ther. 1: 332-337.

Single chain antibodies comprise the variable domains of the heavy andlight chain joined by a flexible linker polypeptide, and are expressedas a single polypeptide. Optionally, single chain antibodies areexpressed as a single chain variable region fragment joined to the lightchain constant region. Well known intracellular trafficking signals areengineered into recombinant polynucleotide vectors encoding such singlechain antibodies in order to precisely target the expressed intrabody tothe desired intracellular compartment. For example, intrabodies targetedto the endoplasmic reticulum (ER) are engineered to incorporate a leaderpeptide and, optionally, a C-terminal ER retention signal, such as theKDEL amino acid motif. Intrabodies intended to exert activity in thenucleus are engineered to include a nuclear localization signal. Lipidmoieties are joined to intrabodies in order to tether the intrabody tothe cytosolic side of the plasma membrane. Intrabodies can also betargeted to exert function in the cytosol. For example, cytosolicintrabodies are used to sequester factors within the cytosol, therebypreventing them from being transported to their natural cellulardestination.

In one embodiment, intrabodies are used to capture 84P2A9 in thenucleus, thereby preventing its activity within the nucleus. Nucleartargeting signals are engineered into such 84P2A9 intrabodies in orderto achieve the desired targeting. Such 84P2A9 intrabodies are designedto bind specifically to a particular 84P2A9 domain. In anotherembodiment, cytosolic intrabodies that specifically bind to the 84P2A9protein are used to prevent 84P2A9 from gaining access to the nucleus,thereby preventing it from exerting any biological activity within thenucleus (e.g., preventing 84P2A9 from forming transcription complexeswith other factors).

In order to specifically direct the expression of such intrabodies toparticular cells, the transcription of the intrabody is placed under theregulatory control of an appropriate tumor-specific promoter and/orenhancer. In order to target intrabody expression specifically toprostate, for example, the PSA promoter and/or promoter/enhancer can beutilized (See, for example, U.S. Pat. No. 5,919,652).

Inhibition of 84P2A9 with Recombinant Proteins

In another approach, recombinant molecules that bind to 84P2A9 therebyprevent or inhibit 84P2A9 from accessing/binding to its bindingpartner(s) or associating with other protein(s) are used to inhibit84P2A9 function. Such recombinant molecules can, for example, containthe reactive part(s) of an 84P2A9 specific antibody molecule. In aparticular embodiment, the 84P2A9 binding domain of an 84P2A9 bindingpartner is engineered into a dimeric fusion protein comprising two84P2A9 ligand binding domains linked to the Fc portion of a human IgG,such as human IgG1. Such IgG portion can contain, for example, the CH2and CH3 domains and the hinge region, but not the CH1 domain. Suchdimeric fusion proteins are administered in soluble form to patientssuffering from a cancer associated with the expression of 84P2A9,including but not limited to prostate and testicular cancers, where thedimeric fusion protein specifically binds to 84P2A9 thereby blocking84P2A9 interaction with a binding partner. Such dimeric fusion proteinsare further combined into multimeric proteins using known antibodylinking technologies.

Inhibition of 84P2A9 Transcription or Translation

Within the second class of therapeutic approaches, the inventionprovides various methods and compositions for inhibiting thetranscription of the 84P2A9 gene. Similarly, the invention also providesmethods and compositions for inhibiting the translation of 84P2A9 mRNAinto protein.

In one approach, a method of inhibiting the transcription of the 84P2A9gene comprises contacting the 84P2A9 gene with an 84P2A9 antisensepolynucleotide. In another approach, a method of inhibiting 84P2A9 mRNAtranslation comprises contacting the 84P2A9 mRNA with an antisensepolynucleotide. In another approach, an 84P2A9 specific ribozyme is usedto cleave the 84P2A9 message, thereby inhibiting translation. Suchantisense and ribozyme based methods can also be directed to theregulatory regions of the 84P2A9 gene, such as the 84P2A9 promoterand/or enhancer elements. Similarly, proteins capable of inhibiting an84P2A9 gene transcription factor can be used to inhibit 84P2A9 mRNAtranscription. The various polynucleotides and compositions useful inthe aforementioned methods have been described above. The use ofantisense and ribozyme molecules to inhibit transcription andtranslation is well known in the art.

Other factors that inhibit the transcription of 84P2A9 throughinterfering with 84P2A9 transcriptional activation are also useful forthe treatment of cancers expressing 84P2A9. Similarly, factors that arecapable of interfering with 84P2A9 processing are useful for thetreatment of cancers expressing 84P2A9. Cancer treatment methodsutilizing such factors are also within the scope of the invention.

General Considerations for Therapeutic Strategies

Gene transfer and gene therapy technologies can be used for deliveringtherapeutic polynucleotide molecules to tumor cells synthesizing 84P2A9(Le., antisense, ribozyme, polynucleotides encoding intrabodies andother 84P2A9 inhibitory molecules). A number of gene therapy approachesare known in the am Recombinant vectors encoding 84P2A9 antisensepolynucleotides, ribozymes, factors capable of interfering with 84P2A9transcription, and so forth, can be delivered to target tumor cellsusing such gene therapy approaches.

The above therapeutic approaches can be combined with any one of a widevariety of surgical chemotherapy or radiation therapy regimens. Thesetherapeutic approaches can enable the use of reduced dosages ofchemotherapy and/or less frequent administration, an advantage for allpatients and particularly for those that do not tolerate the toxicity ofthe chemotherapeutic agent well.

The anti-tumor activity of a particular composition (e.g., antisense,ribozyme, intrabody), or a combination of such compositions, can beevaluated using various in vitro and in vivo assay systems. In vitroassays for evaluating therapeutic potential include cell growth assays,soft agar assays and other assays indicative of tumor promotingactivity, binding assays capable of determining the extent to which atherapeutic composition will inhibit the binding of 84P2A9 to a bindingpartner, etc.

In vivo, the effect of an 84P2A9 therapeutic composition can beevaluated in a suitable animal model. For example, xenogenic prostatecancer models wherein human prostate cancer explants or passagedxenograft tissues are introduced into immune compromised animals, suchas nude or SCID mice, are appropriate in relation to prostate cancer andhave been described (Klein et al., 1997, Nature Medicine 3: 402-408).For example, PCT Patent Application WO98/16628, Sawyers et al.,published Apr. 23, 1998, describes various xenograft models of humanprostate cancer capable of recapitulating the development of primarytumors, micrometastasis, and the formation of osteoblastic metastasescharacteristic of late stage disease. Efficacy can be predicted usingassays that measure inhibition of tumor formation, tumor regression ormetastasis, and the like. See, also, the Examples below.

In vivo assays that evaluate the promotion of apoptosis are useful inevaluating therapeutic compositions. In one embodiment, xenografts fromtumor bearing mice treated with the therapeutic composition can beexamined for the presence of apoptotic foci and compared to untreatedcontrol xenograft-bearing mice. The extent to which apoptotic foci arefound in the tumors of the treated mice provides an indication of thetherapeutic efficacy of the composition.

The therapeutic compositions used in the practice of the foregoingmethods can be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material that when combined with the therapeutic compositionretains the anti-tumor function of the therapeutic composition and isgenerally non-reactive with the patient's immune system. Examplesinclude, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like (see, generally,Remington's Pharmaceutical Sciences 16^(th) Edition, A. Osal., Ed.,1980).

Therapeutic formulations can be solubilized and administered via anyroute capable of delivering the therapeutic composition to the tumorsite. Potentially effective routes of administration include, but arenot limited to, intravenous, parenteral, intraperitoneal, intramuscular,intratumor, intradermal intraorgan, orthotopic, and the like. Apreferred formulation for intravenous injection comprises thetherapeutic composition in a solution of preserved bacteriostatic water,sterile unpreserved water, and/or diluted in polyvinylchloride orpolyethylene bags containing 0.9% sterile Sodium Chloride for Injection,USP. Therapeutic protein preparations can be lyophilized and stored assterile powders, preferably under vacuum, and then reconstituted inbacteriostatic water containing, for example, benzyl alcoholpreservative, or in sterile water prior to injection.

Dosages and administration protocols for the treatment of cancers usingthe foregoing methods will vary with the method and the target cancer,and will generally depend on a number of other factors appreciated inthe art.

Cancer Vaccines

As noted above, the expression profile of 84P2A9 shows that it is highlyexpressed in advanced and metastasized prostate cancer. This expressionpattern is reminiscent of the Cancer-Testis (CT) antigens or MAGEantigens, which are testis-specific genes that are up-regulated inmelanomas and other cancers (Van den Eynde and Boon, Int J Clin Lab Res.27:81-86, 1997). Due to their tissue-specific expression and highexpression levels in cancer, the MAGE antigens are currently beinginvestigated as targets for cancer vaccines (Durrant, Anticancer Drugs8:727-733, 1997; Reynolds et al., Int J Cancer 72:972-976, 1997).

The invention further provides cancer vaccines comprising an84P2A9-related protein or fragment as well as DNA based vaccines. Inview of the expression of 84P2A9, cancer vaccines are effective atspecifically preventing and/or treating 84P2A9 expressing cancerswithout creating non-specific effects on non-target tissues. The use ofa tumor antigen in a vaccine for generating humoral and cell-mediatedimmunity for use in anti-cancer therapy is well known in the art and hasbeen employed in prostate cancer using human PSMA and rodent PAPimmunogens (Hodge et al., 1995, Int. J. Cancer 63:231-237; Fong et al.,1997, J. Immunol. 159:3113-3117).

Such methods can be readily practiced by employing an 84P2A9 protein, orfragment thereof, or an 84P2A9-encoding nucleic acid molecule andrecombinant vectors capable of expressing and appropriately presentingthe 84P2A9 immunogen (which typically comprises a number of humoral or Tcell epitopes immunoreactive epitopes). In this context, skilledartisans understand that a wide variety of different vaccine systems fordelivery of immunoreactive epitopes are known in the art (see, e.g.,Heryln et al., Ann Med 1999 February; 31(1):66-78; Maruyama et al.,Cancer Immunol Immunother 2000 June; 49(3):123-32) Briefly, suchtechniques consists of methods of generating an immune response (e.g. ahumoral and/or cell mediated response) in a mammal comprising the stepsexposing the mammal's immune system to an immunoreactive epitope (e.g.an epitope of the 84P2A9 protein shown in SEQ ID NO: 2) so that themammal generates an immune response that is specific for that epitope(e.g. generates antibodies that specifically recognize that epitope). Ina preferred method, the 84P2A9 immunogen contains a biological motif. Ina highly preferred embodiment, the 84P2A9 immunogen contains one or moreamino acid sequences identified using one of the pertinent analyticaltechniques well known in the art such as the sequences shown in Table 1.

A wide variety of methods for generating an immune response in a mammalare well known in the art (for example as the first step in thegeneration of hybridomas). Methods of generating an immune response in amammal comprise exposing the mammal's immune system to an exogenousimmunogenic epitope on a protein (e.g. the 84P2A9 protein of SEQ ID NO:2) so that an immune response is generated. A typical embodimentconsists of a method for generating an immune response to 84P2A9 in ahost, by contacting the host with a sufficient amount of 84P2A9 or a Bcell or cytotoxic T-cell eliciting epitope or analog thereof; and atleast one periodic interval thereafter contacting the host withadditional 84P2A9 or a B cell or cytotoxic T-cell eliciting epitope oranalog thereof. A specific embodiment consists of a method of generatingan immune response against an 84P2A9 protein or a multiepitopic peptidecomprising administering 84P2A9 immunogen (e.g. the 84P2A9 protein or apeptide fragment thereof, an 84P2A9 fusion protein etc.) in a vaccinepreparation to humans or animals. Typically, such vaccine preparationsfurther contain a suitable adjuvant. (see, e.g., U.S. Pat. No.6,146,635). A representative variation on these methods consists of amethod of generating an immune response in an individual against an84P2A9 immunogen comprising administering in vivo to muscle or skin ofthe individual's body a genetic vaccine facilitator such as one selectedfrom the group consisting of: anionic lipids; saponins; lectins;estrogenic compounds; hydroxylated lower alkyls; dimethyl sulfoxide; andurea; and a DNA molecule that is dissociated from an infectious agentand comprises a DNA sequence that encodes the 84P2A9 immunogen, the DNAsequence operatively linked to regulatory sequences which control theexpression of the DNA sequence; wherein the DNA molecule is taken up bycells, the DNA sequence is expressed in the cells and an immune responseis generated against the immunogen. (see, e.g., U.S. Pat. No.5,962,428).

In an illustrative example of a specific method for generating an immuneresponse, viral gene delivery systems are used to deliver an84P2A9-encoding nucleic acid molecule. Various viral gene deliverysystems that can be used in the practice of this aspect of the inventioninclude, but are not limited to, vaccinia, fowlpox, canarypox,adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus,and sindbus virus (Restifo, 1996, Curr. Opin. Immunol. 8:658-663).Non-viral delivery systems can also be employed by using naked DNAencoding an 84P2A9 protein or fragment thereof introduced into thepatient (e.g., intramuscularly or intradermally) to induce an anti-tumorresponse. In one embodiment, the full-length human 84P2A9 cDNA isemployed. In another embodiment, 84P2A9 nucleic acid molecules encodingspecific cytotoxic T lymphocyte (CTL) epitopes can be employed. CTLepitopes can be determined using specific algorithms (e.g., Epimer,Brown University) to identify peptides within an 84P2A9 protein that arecapable of optimally binding to specified HLA alleles.

Various ex vivo strategies can also be employed. One approach involvesthe use of dendritic cells to present 84P2A9 antigen to a patient'simmune system. Dendritic cells express MHC class I and II molecules, B7co-stimulator, and IL-12, and are thus highly specialized antigenpresenting cells. In prostate cancer, autologous dendritic cells pulsedwith peptides of the prostate-specific membrane antigen (PSMA) are beingused in a Phase I clinical trial to stimulate prostate cancer patients'immune systems (Tjoa et al., 1996, Prostate 28:65-69; Murphy et al.,1996, Prostate 29:371-380). Thus, dendritic cells can be used to present84P2A9 peptides to T cells in the context of MHC class I and IImolecules. In one embodiment, autologous dendritic cells are pulsed with84P2A9 peptides capable of binding to MHC class I and/or class IImolecules. In another embodiment, dendritic cells are pulsed with thecomplete 84P2A9 protein. Yet another, embodiment involves engineeringthe overexpression of the 84P2A9 gene in dendritic cells using variousimplementing vectors known in the art, such as adenovirus (Arthur etal., 1997, Cancer Gene Ther. 4:17-25), retrovirus (Henderson et al.,1996, Cancer Res. 56:3763-3770), lentivirus, adeno-associated virus, DNAtransfection (Ribas et al., 1997, Cancer Res. 57:2865-2869), ortumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med.186:1177-1182). Cells expressing 84P2A9 can also be engineered toexpress immune modulators, such as GM-CSF, and used as immunizingagents.

Anti-idiotypic anti-84P2A9 antibodies can also be used in anti-cancertherapy as a vaccine for inducing an immune response to cells expressingan 84P2A9 protein. Specifically, the generation of anti-idiotypicantibodies is well known in the art and can readily be adapted togenerate anti-idiotypic anti-84P2A9 antibodies that mimic an epitope onan 84P2A9 protein (see, for example, Wagner et al., 1997, Hybridoma 16:33-40; Foon et al., 1995, J. Clin. Invest 96:334-342; Herlyn et al.,1996, Cancer Immunol. Immunother. 43:65-76). Such an anti-idiotypicantibody can be used in cancer vaccine strategies.

Genetic immunization methods can be employed to generate prophylactic ortherapeutic humoral and cellular immune responses directed againstcancer cells expressing 84P2A9. Constructs comprising DNA encoding an84P2A9-related protein/immunogen and appropriate regulatory sequencescan be injected directly into muscle or skin of an individual, such thatthe cells of the muscle or skin take-up the construct and express theencoded 84P2A9 protein/immunogen. Alternatively, a vaccine comprises an84P2A9-related protein. Expression of the 84P2A9 protein immunogenresults in the generation of prophylactic or therapeutic humoral andcellular immunity against bone, colon, pancreatic, testicular, cervicaland ovarian cancers. Various prophylactic and therapeutic geneticimmunization techniques known in the art can be used.

Kits

For use in the diagnostic and therapeutic applications described herein,kits are also provided by the invention. Such kits can comprise acarrier being compartmentalized to receive in close confinement one ormore containers such as vials, tubes, and the like, each of thecontainer(s) comprising one of the separate elements to be used in themethod. For example, the container(s) can comprise a probe that is orcan be detectably labeled. Such probe can be an antibody orpolynucleotide specific for an 84P2A9-related protein or an 84P2A9 geneor message, respectively. Where the kit utilizes nucleic acidhybridization to detect the target nucleic acid, the kit can also havecontainers containing nucleotide(s) for amplification of the targetnucleic acid sequence and/or a container comprising a reporter-means,such as a biotin-binding protein, such as avidin or streptavidin, boundto a reporter molecule, such as an enzymatic, florescent, orradioisotope label.

The kit of the invention will typically comprise the container describedabove and one or more other containers comprising materials desirablefrom a commercial and user standpoint, including buffers, diluents,filters, needles, syringes, and package inserts with instructions foruse. A label can be present on the container to indicate that thecomposition is used for a specific therapy or non-therapeuticapplication, and can also indicate directions for either in vivo or invitro use, such as those described above.

p84P2A9-1 has been deposited under the requirements of the BudapestTreaty for the Deposit of Microorganisms for Patent Purposes on Jan. 6,2000 with the American Type Culture Collection (ATCC), 10801 UniversityBlvd., Manassas, Va. 20110-2209 USA, and has been identified as ATCCAccession No. PTA-1151. All restrictions on access to this deposit willbe irrevocably removed prior to issuance of a patent on the presentapplication or counterpart thereof.

EXAMPLES

Various aspects of the invention are further described and illustratedby way of the several examples that follow, none of which are intendedto limit the scope of the invention.

Example 1 SSH-Generated Isolation of cDNA Fragment of the 84P2A9 Gene

Materials and Methods

LAPC Xenografts and Human Tissues

LAPC xenografts were obtained from Dr. Charles Sawyers (UCLA) andgenerated as described (Klein et al, 1997, Nature Med. 3: 402-408).Androgen dependent and independent LAPC-4 xenografts LAPC-4 AD and AI,respectively) and LAPC-9 AD and AI xenografts were grown in male SCIDmice and were passaged as small tissue chunks in recipient males. LAPC-4and -9 AI xenografts were derived from LAPC-4 or -9 AD tumors,respectively. Male mice bearing AD tumors were castrated and maintainedfor 2-3 months. After the tumors re-grew, the tumors were harvested andpassaged in castrated males or in female SCID mice. Human tissues forRNA and protein analyses were obtained from the Human Tissue ResourceCenter (HTRC) at the UCLA (Los Angeles, Calif.) and from QualTek, Inc.(Santa Barbara, Calif.). A benign prostatic hyperplasia tissue samplewas patient-derived.

Cell Lines

Human cell lines (e.g., HeLa) were obtained from the ATCC and weremaintained in DMEM with 5% fetal calf serum.

RNA Isolation

Tumor tissue and cell lines were homogenized in Trizol reagent (LifeTechnologies, Gibco BRL) using 10 ml/g tissue or 10 ml/10⁸ cells toisolate total RNA. Poly A RNA was purified from total RNA using Qiagen'sOligotex mRNA Mini and Midi kits. Total and mRNA were quantified byspectrophotometric analysis (O.D. 260/280 nm) and analyzed by gelelectrophoresis.

Oligonucleotides

The following HPLC purified oligonucleotides were used:

DPNCDN (cDNA synthesis primer) (SEQ ID NO: 7) 5′TTTTGATCAAGGTT₃₀3′Adaptor 1: (SEQ ID NO: 8) 5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′(SEQ ID NO: 9) 3′GGCCCGTCCTAG5′ Adaptor 2: (SEQ ID NO: 10)5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′ (SEQ ID NO: 11)3′CGGCTCCTAG5′ PCR primer 1: (SEQ ID NO: 12) 5′CTAATACGACTCACTATAGGGC3′Nested primer (NP) 1: (SEQ ID NO: 13) 5′TCGAGCGGCCGCCCGGGCAGGA3′ Nestedprimer (NP)2: (SEQ ID NO: 14) 5′AGCGTGGTCGCGGCCGAGGA3′

Suppression Subtractive Hybridization

Suppression Subtractive Hybridization (SSH) was used to identify cDNAscorresponding to genes that may be differentially expressed in prostatecancer. The SSH reaction utilized cDNA from two LAPC-4 AD xenografts.Specifically, the 84P2A9 SSH sequence was identified from a subtractionwhere cDNA derived from an LAPC-4 AD tumor, 3 days post-castration, wassubtracted from cDNA derived from an LAPC-4 AD tumor grown in an intactmale. The LAPC-4 AD xenograft tumor grown in an intact male was used asthe source of the “tester” cDNA, while the cDNA from the LAPC-4 ADtumor, 3 days post-castration, was used as the source of the “driver”cDNA.

Double stranded cDNAs corresponding to tester and driver cDNAs weresynthesized from 2 μg of poly(A)⁺ RNA isolated from the relevantxenograft tissue, as described above, using CLONTECH's PCR-Select cDNASubtraction Kit and 1 ng of oligonucleotide DPNCDN as primer. First- andsecond-strand synthesis were carried out as described in the Kit's usermanual protocol (CLONTECH Protocol No. PT1117-1, Catalog No. K1804-1).The resulting cDNA was digested with Dpn II for 3 hours at 37° C.Digested cDNA was extracted with phenol/chloroform (1:1) and ethanolprecipitated.

Driver cDNA was generated by combining in a 1:1 ratio Dpn II digestedcDNA from the relevant xenograft source (see above) with a mix ofdigested cDNAs derived from human benign prostatic hyperplasia (BPH),the human cell lines HeLa, 293, A431, Colo205, and mouse liver.

Tester cDNA was generated by diluting 1 μl of Dpn II digested cDNA fromthe relevant xenograft source (see above) (400 ng) in 5 μl of water. Thediluted cDNA (2 μl, 160 ng) was then ligated to 2 μl of Adaptor 1 andAdaptor 2 (10 μM, in separate ligation reactions, in a total volume of10 μl at 16° C. overnight, using 400 u of T4 DNA ligase (CLONTECH).Ligation was terminated with 1 μl of 0.2 M EDTA and heating at 72° C.for 5 min.

The first hybridization was performed by adding 1.5 μl (600 ng) ofdriver cDNA to each of two tubes containing 1.5 μl (20 ng) Adaptor 1-and Adaptor 2-ligated tester cDNA. In a final volume of 4 μl, thesamples were overlaid with mineral oil, denatured in an MJ Researchthermal cycler at 98° C. for 1.5 minutes, and then were allowed tohybridize for 8 hours at 68° C. The two hybridizations were then mixedtogether with an additional 1 μl of fresh denatured driver cDNA and wereallowed to hybridize overnight at 68° C. The second hybridization wasthen diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA,heated at 70° C. for 7 min. and stored at −20° C.

PCR Amplification, Cloning and Sequencing of Gene Fragments Generatedfrom SSH

To amplify gene fragments resulting from SSH reactions, two PCRamplifications were performed. In the primary PCR reaction 1 μl of thediluted final hybridization mix was added to 1 μl of PCR primer 1 (10μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10× reaction buffer (CLONTECH) and0.5 μl 50× Advantage cDNA polymerase Mix (CLONTECH) in a final volume of25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C.for 30 sec, 72° C. for 1.5 min. Five separate primary PCR reactions wereperformed for each experiment. The products were pooled and diluted 1:10with water. For the secondary PCR reaction, 1 μl from the pooled anddiluted primary PCR reaction was added to the same reaction mix as usedfor PCR 1, except that primers NP1 and NP2 (10 μM) were used instead ofPCR primer 1. PCR 2 was performed using 10-12 cycles of 94° C. for 10sec, 68° C. for 30 sec, 72° C. for 1.5 minutes. The PCR products wereanalyzed using 2% agarose gel electrophoresis.

The PCR products were inserted into pCR2.1 using the T/A vector cloningkit (Invitrogen). Transformed E. coli were subjected to blue/white andampicillin selection. White colonies were picked and arrayed into 96well plates and were grown in liquid culture overnight. To identifyinserts, PCR amplification was performed on 1 ml of bacterial cultureusing the conditions of PCR1 and NP1 and NP2 as primers. PCR productswere analyzed using 2% agarose gel electrophoresis.

Bacterial clones were stored in 20% glycerol in a 96 well format PlasmidDNA was prepared, sequenced, and subjected to nucleic acid homologysearches of the GenBank, dbest, and NCI-CGAP databases.

RT-PCR Expression Analysis

First strand cDNAs can be generated from 1 μg of mRNA with oligo(dT)12-18 priming using the Gibco-BRL Superscript Preamplificationsystem. The manufacturer's protocol can be used and included anincubation for 50 min at 42° C. with reverse transcriptase followed byRNAse H treatment at 37° C. for 20 min. After completing the reaction,the volume can be increased to 200 μl with water prior to normalization.First strand cDNAs from 16 different normal human tissues can beobtained from Clontech.

Normalization of the first strand cDNAs from multiple tissues can beperformed by using the primers 5′atatcgccgcgctcgtcgtcgacaa3′ (SEQ ID NO:15) and 5′agccacacgcagctcattgtagaagg 3′ (SEQ ID NO: 16) to amplify.beta.-actin. First strand cDNA (5 μl) can be amplified in a totalvolume of 50 μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1×PCRbuffer (Clontech, 10 mM Tris-HCL, 1.5 mM MgCl₂, 50 mM KCl, pH8.3) and 1×Klentaq DNA polymerase (Clontech). Five μl of the PCR reaction can beremoved at 18, 20, and 22 cycles and used for agarose gelelectrophoresis. PCR can be performed using an MJ Research thermalcycler under the following conditions: Initial denaturation can be at94° C. for 15 sec, followed by a 18, 20, and 22 cycles of 94° C. for 15,65° C. for 2 min, 72° C. for 5 sec. A final extension at 72° C. can becarried out for 2 min. After agarose gel electrophoresis, the bandintensities of the 283 bp .beta.-actin bands from multiple tissues canbe compared by visual inspection. Dilution factors for the first strandcDNAs can be calculated to result in equal .beta.-actin band intensitiesin all tissues after 22 cycles of PCR. Three rounds of normalization canbe required to achieve equal band intensities in all tissues after 22cycles of PCR.

To determine expression levels of the 84P2A9 gene, 5 μl of normalizedfirst strand cDNA can be analyzed by PCR using 25, 30, and 35 cycles ofamplification using primer pairs that can be designed with theassistance of a MIT web site.

Semi quantitative expression analysis can be achieved by comparing thePCR products at cycle numbers that give light band intensities.

Results

Two SSH experiments described in the Materials and Methods, supra, ledto the isolation of numerous candidate gene fragment clones (SSHclones). All candidate clones were sequenced and subjected to homologyanalysis against all sequences in the major public gene and ESTdatabases in order to provide information on the identity of thecorresponding gene and to help guide the decision to analyze aparticular gene for differential expression. In general, gene fragmentsthat had no homology to any known sequence in any of the searcheddatabases, and thus considered to represent novel genes, as well as genefragments showing homology to previously sequenced expressed sequencetags (ESTs), were subjected to differential expression analysis byRT-PCR and/or Northern analysis.

One of the SHH clones comprising about 425 bp, showed significanthomology to several testis-derived ESTs but no homology to any knowngene, and was designated 84P2A9.

Northern expression analysis of first strand cDNAs from 16 normaltissues showed a highly prostate and testis-related expression patternin adult tissues (FIG. 4).

Example 2 Full Length Cloning of 84P2A9

A full length 84P2A9 cDNA clone (clone 1) of 2347 base pairs (bp) wascloned from an LAPC-4 AD cDNA library (Lambda ZAP Express, Stratagene)(FIG. 2). The cDNA encodes an open reading frame (ORF) of 504 aminoacids. Sequence analysis revealed the presence of six potential nuclearlocalization signals and is predicted to be nuclear using the PSORTprogram. The protein sequence is homologous to a human brain proteinKIAA1152 (39.5% identity over a 337 amino acid region), and exhibits adomain that is homologous to the LUCA15 tumor suppressor protein (64.3%identity over a 42 amino acid region)(GenBank Accession #P52756)(FIG.3). The 84P2A9 cDNA was deposited on Jan. 5, 2000 with the American TypeCulture Collection (ATCC; Manassas, Va.) as plasmid p84P2A9-1, and hasbeen assigned Accession No. PTA-1151.

The 84P2A9 proteins have no homology to any known proteins, but thesequence does overlap with several ESTs derived from testis.

Example 3 84P2A9 Gene Expression Analysis

84P2A9 mRNA expression in normal human tissues was analyzed by Northernblotting of two multiple tissue blots (Clontech; Palo Alto, Calif.),comprising a total of 16 different normal human tissues, using labeled84P2A9 SSH fragment (Example 1) as a probe. RNA samples werequantitatively, normalized with a .beta.-actin probe. The resultsdemonstrated expression of a 2.4 and 4.5 kb transcript in normal testisand prostate (FIG. 4).

To analyze 84P2A9 expression in prostate cancer tissues lines northernblotting was performed on RNA derived from the LAPC xenografts. Theresults show high levels of 84P2A9 expression in all the xenografts,with the highest levels detected in LAPC-9 AD, LAPC-9 AI (FIG. 4 andFIG. 5). These results provide evidence that 84P2A9 is up-regulated inprostate cancer.

In addition, high levels of expression were detected in brain (PFSK-1,-T98G), bone (HOS, U2-OS), lung (CALU-1, NCI-H82, NCI-H146), and kidney(769-P, A498, CAKI-1, SW839) cancer cell lines (FIG. 5). Moderateexpression levels were detected in several pancreatic (PANC-1, BxPC-3,HPAC, CAPAN-1), colon (SK-CO-1, CACO-2, LOVO, COLO-205), bone (SK-ES-1,RD-ES), breast (MCF-7, MDA-MB435s) and testicular cancer (NCCIT) celllines (FIG. 5).

In addition, prostate cancer patient samples show expression of 84P2A9in both the normal and the tumor part of the prostate tissues (FIG. 6).These results suggest that 84P2A9 is a very testis specific gene that isup-regulated in prostate cancer and potentially other cancers. Similarto the MAGE antigens, 84P2A9 may thus qualify as a cancer-testis antigen(Van den Eynde and Boon, Int J Clin Lab Res. 27:81-86, 1997).

84P2A9 expression in normal tissues can be further analyzed using amulti-tissue RNA dot blot containing different samples (representingmainly normal tissues as well as a few cancer cell lines).

Example 4 Generation of 84P2A9 Polyclonal Antibodies

Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Typicallya peptide can be designed from a coding region of 84P2A9. Alternativelythe immunizing agent may include all or portions of the 84P2A9 protein,or fusion proteins thereof. For example, the 84P2A9 amino acid sequencecan be fused to any one of a variety of known fusion protein partnersthat are well known in the art such as maltose binding protein, LacZ,thioredoxin or an immunoglobulin constant region (see, e.g., CurrentProtocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubulet al. eds., 1995; Linsley, P. S., Brady, W., Urnes, M., Grosmaire, L.,Damle, N., and Ledbetter, L. (1991) J. Exp. Med. 174, 561-566). Othersuch recombinant bacterial proteins include glutathione-5-transferase(GST), and HIS tagged fusion proteins of 84P2A9 (which can be purifiedfrom induced bacteria using the appropriate affinity matrix).

It may be useful to conjugate the immunizing agent to a protein known tobe immunogenic in the mammal being immunized. Examples of suchimmunogenic proteins include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Examples of adjuvants which may be employed include Freund'scomplete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate).

In a typical protocol, rabbits can be initially immunized subcutaneouslywith about 200 μg of fusion protein or KLH-peptide mixed in completeFreund's adjuvant Rabbits are then injected subcutaneously every twoweeks with about 200 μg of immunogen in incomplete Freund's adjuvant.Test bleeds are taken approximately 7-10 days following eachimmunization and used to monitor the titer of the antiserum by ELISA.

Specificity of the antiserum is tested by Western blot andimmunoprecipitation analyses using lysates of genetically engineeredcells or cells expressing endogenous 84P2A9. To genetically engineercells to express 84P2A9, the full length 84P2A9 cDNA can be cloned intoan expression vector that provides a 6His tag at the carboxyl-terminus(pcDNA 3.1 myc-his, InVitrogen). After transfection of the constructsinto 293T cells, cell lysates can be immunoprecipitated and Westernblotted using anti-His or v5 anti-epitope antibody (Invitrogen) and theanti-84P2A9 serum (see, e.g., FIG. 11). Sera from His-tagged protein andpeptide immunized rabbits as well as depleted GST and MBP protein seraare purified by passage over an affinity column composed of therespective immunogen covalently coupled to Affigel matrix (BioRad).

Example 5 Production of Recombinant 84P2A9 in Bacterial and MammalianSystems

Bacterial Constructs: Production of Recombinant 84P2A9 Using pGEXConstructs

To express 84P2A9 in bacterial cells, a portion of 84P2A9 was fused tothe Glutathione S-transferase (GST) gene by cloning into pGEX-6P-1(Amersham Pharmacia Biotech, N.J.). All constructs were made to generaterecombinant 84P2A9 protein sequences with GST fused at the N-terminusand a six histidine epitope at the C-terminus. The six histidine epitopetag was generated by adding the histidine codons to the cloning primerat the 3′ end of the ORF. A PreScission™ recognition site permitscleavage of the GST tag from 84P2A9. The ampicillin resistance gene andpBR322 origin permits selection and maintenance of the plasmid in E.coli. In this construct, a fragment containing amino acids 1 to 151 of84P2A9 was cloned into pGEX-6P-1. Additional constructs can be made inpGEX-6P-1 spanning regions of the 84P2A9 protein such as amino acids 1to 504 and amino acids 151 to 504.

Mammalian Constructs

To express recombinant 84P2A9 in mammalian systems, the full length84P2A9 cDNA can for example, be cloned into an expression vector thatprovides a 6His tag at the carboxyl-terminus (pCDNA 3.1 myc-his,InVitrogen). The constructs can be transfected into 293T cells.Transfected 293T cell lysates can be probed with the anti-84P2A9polyclonal serum described in Example 4 above in a Western blot.

The 84P2A9 genes can also be subcloned into the retroviral expressionvector pSR.alpha.MSVtkneo and used to establish 84P2A9 expressing celllines as follows. The 84P2A9 coding sequence (from translationinitiation ATG to the termination codons) is amplified by PCR using dscDNA template from 84P2A9 cDNA. The PCR product is subcloned intopSR.alpha.MSVtkneo via the EcoR1 (blunt-ended) and Xba 1 restrictionsites on the vector and transformed into DH5.alpha. competent cells.Colonies are picked to screen for clones with unique internalrestriction sites on the cDNA. The positive clone is confirmed bysequencing of the cDNA insert Retroviruses may thereafter be used forinfection and generation of various cell lines using, for example, NIH3T3, TsuPr1, 293 or rat-1 cells.

Specific mammalian systems are discussed herein.

Production of Recombinant 84P2A9 Using pcDNA3.1/V5-His-TOPO Constructs

To express 84P2A9 in mammalian cells, the 1512 bp (504 amino acid)84P2A9 ORF along with perfect translational start Kozak consensussequence was cloned into pcDNA3.1V5-His-TOPO (Invitrogen, Carlsbad,Calif.). Protein expression is driven from the cytomegalovirus (CMV)promoter. The recombinant protein has the V5 epitope and six histidinesfused to the C-terminus. The pcDNA3.1/V5-His-TOPO vector also containsthe bovine growth hormone (BGH) polyadenylation signal and transcriptiontermination sequence to enhance mRNA stability along with the SV40origin for episomal replication and simple vector rescue in cell linesexpressing the large T antigen. The Neomycin resistance gene allows forselection of mammalian cells expressing the protein and the ampicillinresistance gene and ColE1 origin permits selection and maintenance ofthe plasmid in E. coli.

pSRa Constructs

To generate mammalian cell lines expressing 84P2A9 constitutively, the1551 bp (517 amino acid) ORF is being cloned into pSRa constructs.Amphotropic and ecotropic retroviruses are generated by transfection ofpSRa constructs into the 293T-10A1 packaging line or co-transfection ofpSRa and a helper plasmid ((.phi.quadrature.) in 293 cells,respectively. The retrovirus can be used to infect a variety ofmammalian cell lines, resulting in the integration of the cloned gene,84P2A9, into the host cell-lines. Protein expression is driven from along terminal repeat (LTR). The Neomycin resistance gene allows forselection of mammalian cells expressing the protein and the ampicillinresistance gene and ColE1 origin permits selection and maintenance ofthe plasmid in E. coli. Additional pSRa constructs are being made toproduce both N-terminal and C-terminal GFP and myc/6 HIS fusion proteinsof the fall-length 84P2A9 protein.

Example 6 Production of Recombinant 84P2A9 in a Baculovirus System

To generate a recombinant 84P2A9 protein in a baculovirus expressionsystem, the 84P2A9 cDNA is cloned into the baculovirus transfer vectorpBlueBac 4.5 (Invitrogen), which provides a His-tag at the N-terminusSpecifically, pBlueBac-84P2A9 is co-transfected with helper plasmidpBac-N-Blue (Invitrogen) into SF9 (Spodoptera frugiperda) insect cellsto generate recombinant baculovirus (see Invitrogen instruction manualfor details). Baculovirus is then collected from cell supernatant andpurified by plaque assay.

Recombinant 84P2A9 protein is then generated by infection of HighFiveinsect cells (InVitrogen) with the purified baculovirus. Recombinant84P2A9 protein may be detected using anti-84P2A9 antibody. 84P2A9protein may be purified and used in various cell based assays or asimmunogen to generate polyclonal and monoclonal antibodies specific for84P2A9.

Example 7 Chromosomal Mapping of the 84P2A9 Gene

The chromosomal localization of 84P2A9 was determined using theGeneBridge4 radiation hybrid panel (Walter et al., 1994, Nat. Genetics7:22) (Research Genetics, Huntsville Ala.). The following PCR primerswere used to localize 84P2A9:

(SEQ ID NO: 17) 84P2A9.1 gacttcactgatgcgatggtaggt (SEQ ID NO: 18)84P2A9.2 gtcaatactttccgatgctttgct

The resulting mapping vector for the 93 radiation hybrid panel DNAs was:00001000110010110010000011000100100100001000101001100010000000100100-1001010000000100000010000. The gene for 84P2A9 was mapped to chromosome1q32.3 (D1S1602-D1S217).

Example 8 Identification of Potential Signal Transduction Pathways

To determine whether 84P2A9 directly or indirectly activates knownsignal transduction pathways in cells, luciferase auc) basedtranscriptional reporter assays are carried out in cells expressing84P2A9. These transcriptional reporters contain consensus binding sitesfor known transcription factors that lie downstream of wellcharacterized signal transduction pathways. The reporters and examplesof there associated transcription factors, signal transduction pathways,and activation stimuli are listed below.

1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress

2. SRE-luc, SRF/TCF/ELK1; MIAPK/SAPK; growth/differentiation

3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress

4. ARE-luc, androgen receptor; steroids/MAPK;growth/differentiation/apoptosis

5. p53-luc, p53; SAPK; growth/differentiation/apoptosis

6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress

84P2A9-mediated effects may be assayed in cells showing mRNA expression.Luciferase reporter plasmids may be introduced by lipid mediatedtransfection (TFX-50, Promega). Luciferase activity, an indicator ofrelative transcriptional activity, is measured by incubation of cellsextracts with luciferin substrate and luminescence of the reaction ismonitored in a luminometer.

Example 9 Generation of 84P2A9 Monoclonal Antibodies

To generate MAbs to 84P2A9, typically Balb C mice are immunizedintraperitoneally with about 10-50 μg of protein immunogen mixed incomplete Freund's adjuvant. Protein immunogens include bacterial andbaculovirus produced recombinant 84P2A9 proteins and mammalian expressedhuman IgG FC fusion proteins. Mice are then subsequently immunized every2-4 weeks with 10-50 μg of antigen mixed in Freund's incompleteadjuvant. Alternatively, Ribi adjuvant is used for initialimmunizations. In addition, a DNA-based immunization protocol is used inwhich a mammalian expression vector such as pcDNA 3.1 encoding the84P2A9 cDNA alone or as an IgG FC fusion is used to immunize mice bydirect injection of the plasmid DNA. This protocol is used alone and incombination with protein immunogens. Test bleeds are taken 7-10following immunization to monitor titer and specificity of the immuneresponse. Once appropriate reactivity and specificity is obtained asdetermined by ELISA, Western blotting, and immunoprecipitation analyses,fusion and hybridoma generation is then carried with establishedprocedures well known in the art (Harlow and Lane, 1988).

In a typical specific protocol, a glutathione-5-transferase (GST) fusionprotein encompassing an 84P2A9 protein is synthesized and used asimmunogen. Balb C mice are initially immunized intraperitoneally with10-50 μg of the GST-84P2A9 fusion protein mixed in complete Freund'sadjuvant Mice are subsequently immunized every 2 weeks with 10-50 μg ofGST-84P2A9 protein mixed in Freund's incomplete adjuvant for a total of3 immunizations. Reactivity of serum from immunized mice to full length84P2A9 protein is monitored by ELISA using a partially purifiedpreparation of HIS-tagged 84P2A9 protein expressed from 293T cellsExample 5). Mice showing the strongest reactivity are rested for 3 weeksand given a final injection of fusion protein in PBS and then sacrificed4 days later. The spleens of the sacrificed mice are then harvested andfused to SPO/2 myeloma cells using standard procedures (Harlow and Lane,1988). Supernatants from growth wells following HAT selection arescreened by ELISA and Western blot to identify 84P2A9 specific antibodyproducing clones.

The binding affinity of an 84P2A9 monoclonal antibody may be determinedusing standard technology. Affinity measurements quantify the strengthof antibody to epitope binding and may be used to help define which84P2A9 monoclonal antibodies are preferred for diagnostic or therapeuticuse. The BIAcore system (Uppsala, Sweden) is a preferred method fordetermining binding affinity. The BIAcore system uses surface plasmonresonance (SPR, Welford K 1991, Opt. Quant. Elect. 23:1; Morton andMyszka, 1998, Methods in Enzymology 295: 268) to monitor biomolecularinteractions in real time. BIAcore analysis conveniently generatesassociation rate constants, dissociation rate constants, equilibriumdissociation constants, and affinity constants.

Example 10 In Vitro Assays of 84P2A9 Function

The expression of 84P2A9 in prostate cancer provides evidence that thisgene has a functional role in tumor progression. It is possible that84P2A9 functions as a transcription factor involved in activating genesinvolved in tumorigenesis or repressing genes that block tumorigenesis.84P2A9 function can be assessed in mammalian cells using in vitroapproaches. For mammalian expression, 84P2A9 can be cloned into a numberof appropriate vectors, including pcDNA 3.1 myc-His-tag (Example 5) andthe retroviral vector pSR.alpha.tkneo (Muller et al., 1991, MCB11:1785). Using such expression vectors, 84P2A9 can be expressed inseveral cell lines, including NIH 3T3, rat-1, TsuPr1 and 293T.Expression of 84P2A9 can be monitored using anti-84P2A9 antibodies (seeExamples 4 and 9).

Mammalian cell lines expressing 84P2A9 can be tested in several in vitroand in vivo assays, including cell proliferation in tissue culture,activation of apoptotic signals, tumor formation in SCID mice, and invitro invasion using a membrane invasion culture system (MICS) (Welch etal., Int. J. Cancer 43: 449-457). 84P2A9 cell phenotype is compared tothe phenotype of cells that lack expression of 84P2A9. Thetranscriptional effect of 84P2A9 can be tested by evaluating the effectof 84P2A9 on gene expression using gene arrays (Clontech) andtranscriptional reporter assays (Stratagene).

Cell lines expressing 84P2A9 can also be assayed for alteration ofinvasive and migratory properties by measuring passage of cells througha matrigel coated porous membrane chamber (Becton Dickinson). Passage ofcells through the membrane to the opposite side is monitored using afluorescent assay (Becton Dickinson Technical Bulletin #428) usingcalcein-Am (Molecular Probes) loaded indicator cells. Cell linesanalyzed include parental and 84P2A9 overexpressing PC3, NIH 3T3 andLNCaP cells. To determine whether 84P2A9-expressing cells havechemoattractant properties, indicator cells are monitored for passagethrough the porous membrane toward a gradient of 84P2A9 conditionedmedia compared to control media. This assay may also be used to qualifyand quantify specific neutralization of the 84P2A9 induced effect bycandidate cancer therapeutic compositions.

The function of 84P2A9 can be evaluated using anti-sense RNA technologycoupled to the various functional assays described above, e.g. growth,invasion and migration. Anti-sense RNA oligonucleotides can beintroduced into 84P2A9 expressing cells, thereby preventing theexpression of 84P2A9. Control and anti-sense containing cells can beanalyzed for proliferation, invasion, migration, apoptotic andtranscriptional potential. The local as well as systemic effect of theloss of 84P2A9 expression can be evaluated.

Example 11 In Vivo Assay for 84P2A9 Tumor Growth Promotion

The effect of the 84P2A9 protein on tumor cell growth may be evaluatedin vivo by gene overexpression in tumor-bearing mice. For example, SCIDmice can be injected SQ on each flank with 1×10⁶ of either PC3, TSUPR1,or DU145 cells containing tkNeo empty vector or 84P2A9. At least twostrategies may be used: (1) Constitutive 84P2A9 expression underregulation of a promoter such as a constitutive promoter obtained fromthe genomes of viruses such as polyoma virus, fowlpox virus (UK2,211,504 published 5 Jul. 1989), adenovirus (such as Adenovirus 2),bovine papilloma virus, avian sarcoma virus, cytomegalovirus, aretrovirus, hepatitis-B virus and Simian Virus 40 (SV40), or fromheterologous mammalian promoters, e.g., the actin promoter or animmunoglobulin promoter, provided such promoters are compatible with thehost cell systems, and (2) Regulated expression under control of aninducible vector system, such as ecdysone, tet, etc., provided suchpromoters are compatible with the host cell systems. Tumor volume isthen monitored at the appearance of palpable tumors and followed overtime to determine if 84P2A9 expressing cells grow at a faster rate andwhether tumors produced by 84P2A9-expressing cells demonstratecharacteristics of altered aggressiveness (e.g. enhanced metastasis,vasculation, reduced responsiveness to chemotherapeutic drugs).Additionally, mice may be implanted with 1×10⁵ of the same cellsorthotopically to determine if 84P2A9 has an effect on local growth inthe prostate or on the ability of the cells to metastasize, specificallyto lungs, lymph nodes, and bone marrow.

The assay is also useful to determine the 84P2A9 inhibitory effect ofcandidate therapeutic compositions, such as for example, 84P2A9intrabodies, 84P2A9 antisense molecules and ribozymes.

Example 12 Western Analysis of 84P2A9 Expression in SubcellularFractions

Sequence analysis of 84P2A9 revealed the presence of nuclearlocalization signal. The cellular location of 84P2A9 can be assessedusing subcellular fractionation techniques widely used in cellularbiology (Storrie B. et al. Methods Enzymol. 1990; 182:203-25). Prostateor testis cell lines can be separated into nuclear, cytosolic andmembrane fractions. The expression of 84P2A9 in the different fractionscan be tested using Western blotting techniques.

Alternatively, to determine the subcellular localization of 84P2A9, 293Tcells can be transfected with an expression vector encoding HIS-tagged84P2A9 (PCDNA 3.1 MYC/HIS, Invitrogen). The transfected cells can beharvested and subjected to a differential subcellular fractionationprotocol as previously described (Pemberton, P. A. et al, 1997, J ofHistochemistry and Cytochemistry, 45:1697-1706.) This protocol separatesthe cell into fractions enriched for nuclei, heavy membranes (lysosomes,peroxisomes, and mitochondria), light membranes (plasma membrane andendoplasmic reticulum), and soluble proteins.

Throughout this application, various publications are referenced (withinparentheses for example). The disclosures of these publications arehereby incorporated by reference herein in their entireties.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any that are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

TABLE 1 predicted binding of peptides from 84P2A9 proteins to the humanMHC class I molecule HLA-A2 Score Start (Estimate of half time) RankPosition Subsequence Residue Listing of disassociation) 1 300 SILTGSFPL(SEQ ID NO: 19) 63.04 2 449 RMLQNMGWT (SEQ ID NO: 20) 33.75 3 4LVHDLVSAL (SEQ ID NO: 21) 29.97 4 238 SLSSTDAGL (SEQ ID NO: 22) 21.36 5198 KIQDEGVVL (SEQ ID NO: 23) 17.28 6 433 FVGENAQPI (SEQ ID NO: 24)17.22 7 301 ILTGSFPLM (SEQ ID NO: 25) 16.05 8 218 KMECEEQKV (SEQ ID NO:26) 11.25 9 480 KGLGLGFPL (SEQ ID NO: 27) 10.47 10 461 GLGRDGKGI (SEQ IDNO: 28) 10.43

TABLES 3-16 provide additional analyses of the predicted binding ofpeptides from 84P2A9 proteins to various HLA molecules.

TABLE 2 AMINO ACID SUBSTITUTION MATRIX A C D E F G H I K L M N P Q R S TV W Y 4 0 −2 −1 −2 0 −2 −1 −1 −1 −1 −2 −1 −1 −1 1 0 0 −3 −2 A 9 −3 −4 −2−3 −3 −1 −3 −1 −1 −3 −3 −3 −3 −1 −1 −1 −2 −2 C 6 2 −3 −1 −1 −3 −1 −4 −31 −1 0 −2 0 −1 −3 −4 −3 D 5 −3 −2 0 −3 1 −3 −2 0 −1 2 0 0 −1 −2 −3 −2 E6 −3 −1 0 −3 0 0 −3 −4 −3 −3 −2 −2 −1 1 3 F 6 −2 −4 −2 −4 −3 0 −2 −2 −20 −2 −3 −2 −3 G 8 −3 −1 −3 −2 1 −2 0 0 −1 −2 −3 −2 2 H 4 −3 2 1 −3 −3 −3−3 −2 −1 3 −3 −1 I 5 −2 −1 0 −1 1 2 0 −1 −2 −3 −2 K 4 2 −3 −3 −2 −2 −2−1 1 −2 −1 L 5 −2 −2 0 −1 −1 −1 1 −1 −1 M 6 −2 0 0 1 0 −3 −4 −2 N 7 −1−2 −1 −1 −2 −4 −3 P 5 1 0 −1 −2 −2 −1 Q 5 −1 −1 −3 −3 −2 R 4 1 −2 −3 −2S 5 0 −2 −2 T 4 −3 −1 V 11 2 W 7 Y Adapted from the GCG Software 9.0BLOSUM62 amino acid substitution matrix (block substitution matrix). Thehigher the value, the more likely a substitution is found in related,natural proteins.

TABLE 3A HLA Peptide Motif Search Results User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested 30 HLA molecule type selected A1 lengthselected for subsequences to be scored 9 echoing mode selected for inputsequence Y echoing format Numbered lines length of user's input peptidesequence 504 number of subsequence scores calculated 496 number oftop-scoring subsequences reported 30 back in scoring output table

TABLE 3B HLA Peptide Scoring Results - 84P2A9 - A19-mers SusequenceScore (Estimate of Half Start Residue Time of Disassociation of AMolecule Rank Position Listing Containing This Subsequence) 1 71SLEEPSKDY 45.000 2 469 ISEPIQAMQ 27.000 3 283 KEDPTELDK 25.000 4 15SSEQARGGF 13.500 5 23 FAETGDHSR 9.000 6 441 ILENNIGNR 9.000 7 241STDAGLFTN 6.250 8 72 LEEPSKDYR 4.500 9 233 ESDSSSLSS 3.750 10 92DSDDQMLVA 3.750 11 157 MTQPPEGCR 2.500 12 413 TGDIKRRRK 2.500 13 256DDEQSDWFY 2.250 14 373 RTEHDQHQL 2.250 15 309 MSHPSRRGF 1.500 16 207ESEETNQTN 1.350 17 231 MSESDSSSL 1.350 18 64 LSEGSDSSL 1.350 19 456WTPGSGLGR 1.250 20 375 EHDQHQLLR 1.250 21 293 VPDPVFESI 1.250 22 93SDDQMLVAK 1.000 23 494 ATTTPNAGK 1.000 24 208 SEETNQTNK 0.900 25 205VLESEETNQ 0.900 26 79 YRENHNNNK 0.900 27 11 ALEESSEQA 0.900 28 226VSDELMSES 0.750 29 31 RSISCPLKR 0.750 30 90 HSDSDDQML 0.750 Echoed UserPeptide Sequence (length = 504 residues) (SEQ ID NO: 2)   1 MEELVHDLVSALEESSEQAR GGFAETGDHS RSISCPLKRQ ARKRRGRKRR  51 SYNVHHPWET GHCLSEGSDSSLEEPSKDYR ENHNNNKKDH SDSDDQMLVA 101 KRRPSSNLNN NVRGKRPLWH ESDFAVDNVGNRTLRRRRKV KRMAVDLPQD 151 ISNKRTMTQP PEGCRDQDMD SDRAYQYQEF TKNKVKKRKLKIIRQGPKIQ 201 DEGVVLESEE TNQTNKDKME CEEQKVSDEL MSESDSSSLS STDAGLFTND251 EGRQGDDEQS DWFYEKESGG ACGITGVVPW WEKEDPTELD KNVPDPVFES 301ILTGSFPLMS HPSRRGFQAR LSRLHGMSSK NIKKSGGTPT SMVPIPGPVG 351 NKRMVHFSPDSHHHDHWFSP GARTEHDQHQ LLRDNRAERG HKKNCSVRTA 401 SRQTSMHLGS LCTGDIKRRRKAAPLPGPTT ACFVGENAQP ILENNIGNRM 451 LQNMGWTPGS GLGRDGKGIS EPIQANQRPKGLGLGFPLPK STSATTTPNA 501 GKSA Each peptide is a portion of SEQ ID NO:2; each start position is specified, the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight.

TABLE 4A HLA Peptide Motif Search Results User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested 30 HLA molecule type selected A1 lengthselected for subsequences to be scored 10 echoing mode selected forinput sequence Y echoing format Numbered lines length of user's inputpeptide sequence 504 number of subsequence scores calculated 495 numberof top-scoring subsequences reported 30 back in scoring output table

TABLE 4B HLA Peptide Scoring Results - 84P2A9 - A1 10-mers SubsequenceScore (Estimate of Half Start Residue Time of Disassociation of aMolecule Rank Position Listing Containing This Subsequence) 1 469ISEPIQAMQR 675.000 2 92 DSDDQMLVAK 30.000 3 207 ESEETNQTNK 27.000 4 168DMDSDRAYQY 25.000 5 11 ALEESSEQAR 9.000 6 71 SLEEPSKDYR 9.000 7 282EKEDPTELDK 4.500 8 166 DQDMDSDRAY 3.750 9 90 HSDSDDQMLV 3.750 10 177YQEFTKNKVK 2.700 11 144 AVDLPQDISN 2.500 12 373 RTEHDQHQLL 2.250 13 33ISCPLKRQAR 1.500 14 231 MSESDSSSLS 1.350 15 15 SSEQARGGFA 1.350 16 254QGDDEQSDWF 1.250 17 255 GDDEQSDWFY 1.250 18 293 VPDPVFESIL 1.250 19 173RAYQYQEFTK 1.000 20 481 GLGLGFPLPK 1.000 21 205 VLESEETNQT 0.900 22 79YRENHNNNKK 0.900 23 441 ILENNIGNRM 0.900 24 23 FAETGDHSRS 0.900 25 121ESDFAVDNVG 0.750 26 233 ESDSSSLSST 0.750 27 409 GSLCTGDIKR 0.750 28 259QSDWFYEKES 0.750 29 70 SSLEEPSKDY 0.750 30 67 GSDSSLEEPS 0.750 EchoedUser Peptide Sequence (length = 504 residues) (SEQ ID NO:2)   1MEELVHDLVS ALEESSEQAR GGFAETGDHS RSISCPLKRQ ARKRRGRKRR  51 SYNVHHPWETGHCLSEGSDS SLEEPSKDYR ENHNNNKKDH SDSDDQMLVA 101 KRRPSSNLNN NVRGKRPLWHESDFAVDNVG NRTLRRRRKV KRMAVDLPQD 151 ISNKRTMTQP PEGCRDQDMD SDRAYQYQEFTKNKVKKRKL KIIRQGPKIQ 201 DEGVVLESEE TNQTNKDKME CEEQKVSDEL MSESDSSSLSSTDAGLFTND 251 EGRQGDDEQS DWFYEKESGG ACGITGVVPW WEKEDPTELD KNVPDPVFES301 ILTGSFPLMS HPSRRGFQAR LSRLHGMSSK NIKKSGGTPT SMVPIPGPVG 351NKRMVHFSPD SHHHDHWFSP GARTEHDQHQ LLRDNRAERG HKKNCSVRTA 401 SRQTSMHLGSLCTGDIKRRR KAAPLPGPTT AGFVGENAQP ILENNIGNRM 451 LQNMGWTPGS GLGRDGKGISEPIQAMQRPK GLGLGFPLPK STSATTTPNA 501 GKSA Each peptide is a portion ofSEQ ID NO: 2; each start position is specified, the length of peptide is10 amino acids, and the end position for each peptide is the startposition plus nine.

TABLE 5A HLA Peptide Motif Search Results User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested 30 HLA molecule type selected A_0201 lengthselected for subsequences to be scored 9 echoing mode selected for inputsequence Y echoing format Numbered lines length of user's input peptidesequence 504 number of subsequence scores calculated 496 number oftop-scoring subsequences reported 30 back in scoring output table

TABLE 5B HLA Peptide Scoring Results - 84P2A9 - A2 9-mers ScoringResults Subsequence Score (Estimate of Half Start Residue Time ofDisassociation of a Molecule Rank Position Listing Containing ThisSubsequence) 1 300 SILTGSFPL 63.035 2 449 RMLQNMGWT 32.748 3 4 LVHDLVSAL29.965 4 238 SLSSTDAGL 21.362 5 198 KIQDEGVVL 17.282 6 433 FVGENAQPI17.217 7 301 ILTGSFPLM 16.047 8 218 KMECEEQKV 11.252 9 480 KGLGLGFPL10.474 10 461 GLGRDGKGI 10.433 11 341 SMVPIPGPV 6.530 12 468 GISEPIQAM6.442 13 405 SMHLGSLCT 5.382 14 191 KIIRQGPKI 5.021 15 117 PLWHESDFA2.445 16 177 YQEFTKNKV 2.076 17 454 MGWTPGSGL 1.968 18 156 TMTQPPEGC1.758 19 374 TEHDQHQLL 1.703 20 52 YNVHHPWET 1.678 21 474 QAMQRPKGL1.098 22 240 SSTDAGLFT 1.097 23 438 AQPILENNI 1.058 24 269 GGACGITGV1.044 25 143 MAVDLPQDI 1.010 26 206 LESEETNQT 1.010 27 173 RAYQYQEFT0.893 28 3 ELVHDLVSA 0.857 29 132 RTLRRRRKV 0.715 30 266 KESGGACGI 0.710Echoed User Peptide Sequence (length = 504 residues) (SEQ ID NO:2)   1MEELVHDLVS ALEESSEQAR GGFAETGDHS RSISCPLKRQ ARKRRGRKRR  51 SYNVHHPWETGHCLSEGSDS SLEEPSKDYR ENHNNNKKDH SDSDDQMLVA 101 KRRPSSNLNN NVRGKRPLWHESDFAVDNVG NRTLRRRRKV KRMAVDLPQD 151 ISNKRTMTQP PEGCRDQDMD SDRAYQYQEFTKNKVKKRKL KIIRQGPKIQ 201 DEGVVLESEE TNQTNKDKME CEEQKVSDEL MSESDSSSLSSTDAGLFTND 251 EGRQGDDEQS DWFYEKESGG AGGITGVVPW WEKEDPTELD KNVPDPVFES301 ILTGSFPLMS HPSRRGFQAR LSRLHGMSSK NIKKSGGTPT SMVPIPGPVG 351NKRMVHFSPD SHHHDHWFSP GARTEHDQHQ LLRDNRAERG HKKNCSVRTA 401 SRQTSMHLGSLCTGDIKRRR KAAPLPGPTT AGFVGENAQP ILENNIGNRM 451 LQNMGWTPGS GLGRDGKGISEPIQAMQRPK GLGLGFPLPK STSATTTPNA 501 GKSA Each peptide is a portion ofSEQ ID NO: 2; each start position is specified, the length of peptide is9 amino acids, and the end position for each peptide is the startposition plus eight.

TABLE 6A HLA Peptide Motif Search Results User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested 30 HLA molecule type selected A_0201 lengthselected for subsequences to be scored 10 echoing mode selected forinput sequence Y echoing format Numbered lines length of user's inputpeptide sequence 504 number of subsequence scores calculated 495 numberof top-scoring subsequences reported 30 back in scoring output table

TABLE 6B HLA Peptide Scoring Results - 84P2A9 - A2 10-mers ScoringResults Subsequence Score (Estimate of Half Start Residue Time ofDisassociation of a Molecule Rank Position Listing Containing ThisSubsequence) 1 230 LMSESDSSSL 107.536 1 230 LMSESDSSSL 107.536 2 63CLSEGSDSSL 87.586 3 117 PLWHESDFAV 73.661 4 453 NMGWTPGSGL 15.428 5 475AMQRPKGLGL 15.428 6 433 FVGENAQPIL 14.454 7 323 RLHGMSSKNI 10.433 8 142RMAVDLPQDI 7.535 9 483 GLGFPLPKST 7.452 10 300 SILTGSFPLM 4.802 11 3ELVHDLVSAL 3.685 12 473 IQAMQRPKGL 3.682 13 292 NVPDPVFESI 3.485 14 124FAVDNVGNRT 1.952 15 334 KSGGTPTSMV 1.589 16 445 NIGNRMLQNM 1.571 17 315RGFQARLSRL 1.187 18 268 SGGACGITGV 1.044 19 288 ELDKNVPDPV 1.022 20 486FPLPKSTSAT 0.828 21 205 VLESEETNQT 0.811 22 402 RQTSMHLGSL 0.648 23 425LPGPTTAGFV 0.552 24 441 ILENNIGNRM 0.541 25 237 SSLSSTDAGL 0.516 26 10SALEESSEQA 0.513 27 212 NQTNKDKMEC 0.504 28 301 ILTGSFPLMS 0.481 29 239LSSTDAGLFT 0.455 30 103 RPSSNLNNNV 0.454 Echoed User Peptide Sequence(length = 504 residues) (SEQ ID NO:2)   1 MEELVHDLVS ALEESSEQARGGFAETGDHS RSISCPLKRQ ARKRRGRKRR  51 SYNVHHPWET GHCLSEGSDS SLEEPSKDYRENHNNNKKDH SDSDDQMLVA 101 KRRPSSNLNN NVRGKRPLWH ESDFAVDNVG NRTLRRRRKVKRMAVDLPQD 151 ISNKRTMTQP PEGCRDQDMD SDRAYQYQEF TKNKVKKRKL KIIRQGPKIQ201 DEGVVLESEE TNQTNKDKME CEEQKVSDEL MSESDSSSLS STDAGLFTND 251EGRQGDDEQS DWFYEKESGG ACGITGVVPW WEKEDPTELD KNVPDPVFES 301 ILTGSFPLMSHPSRRGFQAR LSRLHGMSSK NIKKSGGTPT SMVPIPGPVG 351 NKRMVHFSPD SHHHDHWFSPGARTEHDQHQ LLRDNRAERG HKKNCSVRTA 401 SRQTSMHLGS LCTGDIKRRR KAAPLPGPTTAGFVGENAQP ILENNIGNRM 451 LQNMGWTPGS GLGRDGKGIS EPIQAMQRPK GLGLGFPLPKSTSATTTPNA 501 GKSA Each peptide is a portion of SEQ ID NO: 2; eachstart position is specified, the length of peptide is 10 amino acids,and the end position for each peptide is the start position plus nine.

TABLE 7A HLA Peptide Motif Search Results User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested 30 HLA molecule type selected A3 lengthselected for subsequences to be scored 9 echoing mode selected for inputsequence Y echoing format Numbered lines length of user's input peptidesequence 504 number of subsequence scores calculated 496 number oftop-scoring subsequences reported 30 back in scoring output table

TABLE 7B HLA Peptide Scoring Results - 84P2A9 - A3 9-mers ScoringResults Subsequence Score (Estimate of Half Start Residue Time ofDisassociation of a Molecule Rank Position Listing Containing ThisSubsequence) 1 326 GMSSKNIKK 120.000 2 245 GLFTNDEGR 60.000 3 133TLRRRRKVK 10.000 4 146 DLPQDISNK 9.000 5 410 SLCTGDIKR 8.000 6 107NLNNNVRGK 6.000 7 258 EQSDWFYEK 4.860 8 71 SLEEPSKDY 4.500 9 381LLRDNRAER 4.000 10 441 ILENNIGNR 1.800 11 494 ATTTPNAGK 1.500 12 301ILTGSFPLM 0.900 13 461 GLGRDGKGI 0.900 14 128 NVGNRTLRR 0.800 15 238SLSSTDAGL 0.600 16 307 PLMSHPSRR 0.600 17 456 WTPGSGLGR 0.600 18 218KMECEEQKV 0.600 19 283 KEDPTELDK 0.540 20 409 GSLCTGDIK 0.450 21 273GITGVVPWW 0.405 22 344 PIPGPVGNK 0.405 23 184 KVKKRKLKI 0.360 24 156TMTQPPEGC 0.300 25 11 ALEESSEQA 0.300 26 180 FTKNKVKKR 0.300 27 35CPLKRQARK 0.300 28 459 GSGLGRDGK 0.300 29 191 KIIRQGPKI 0.270 30 483GLGFPLPKS 0.270 Echoed User Peptide Sequence (length = 504 residues)(SEQ ID NO:2)   1 MEELVHDLVS ALEESSEQAR GGFAETGDHS RSISCPLKRQ ARKRRGRKRR 51 SYNVHHPWET GHCLSEGSDS SLEEPSKDYR ENHNNNKKDH SDSDDQMLVA 101KRRPSSNLNN NVRGKRPLWH ESDFAVDNVG NRTLRRRRKV KRMAVDLPQD 151 ISNKRTMTQPPEGCRDQDMD SDRAYQYQEF TKNKVKKRKL KIIRQGPKIQ 201 DEGVVLESEE TNQTNKDKMECEEQKVSDEL MSESDSSSLS STDAGLFTND 251 EGRQGDDEQS DWFYEKESGG ACGITGVVPWWEKEDPTELD KNVPDPVFES 301 ILTGSFPLMS HPSRRGFQAR LSRLHGMSSK NIKKSGGTPTSMVPIPGPVG 351 NKRMVHFSPD SHHHDHWFSP GARTEHDQHQ LLRDNRAERG HKKNCSVRTA401 SRQTSMHLGS LCTGDIKRRR KAAPLPGPTT AGFVGENAQP ILENNIGNRM 451LQNMGWTPGS GLGRDGKGIS EPIQAMQRPK GLGLGFPLPK STSATTTPNA 501 GKSA Eachpeptide is a portion of SEQ ID NO: 2; each start position is specified,the length of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight.

TABLE 8A HLA Peptide Motif Search Results User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested 30 HLA molecule type selected A3 lengthselected for subsequences to be scored 10 echoing mode selected forinput sequence Y echoing format Numbered lines length of user's inputpeptide sequence 504 number of subsequence scores calculated 495 numberof top-scoring subsequences reported 30 back in scoring output table

TABLE 8B HLA Peptide Scoring Results - 84P2A9 - A3 10-mers ScoringResults Subsequence Score (Estimate of Half Start Residue Time ofDisassociation of a Molecule Rank Position Listing Containing ThisSubsequence) 1 481 GLGLGFPLPK 360.000 2 189 KLKIIRQGPK 18.000 3 71SLEEPSKDYR 6.000 4 11 ALEESSEQAR 6.000 5 380 QLLRDNRAER 6.000 6 175YQYQEFTKNK 4.500 7 274 ITGVVPWWEK 4.500 8 133 TLRRRRKVKR 4.000 9 168DMDSDRAYQY 3.600 10 173 RAYQYQEFTK 3.000 11 410 SLCTGDIKRR 3.000 12 156TMTQPPEGCR 1.800 13 146 DLPQDISNKR 1.800 14 107 NLNNNVRGKR 1.800 15 475AMQRPKGLGL 1.200 16 63 CLSEGSDSSL 0.900 17 453 NMGWTPGSGL 0.900 18 230LMSESDSSSL 0.900 19 3 ELVHDLVSAL 0.810 20 132 RTLRRRRKVK 0.750 21 180FTKNKVKKRK 0.750 22 343 VPIPGPVGNK 0.608 23 238 SLSSTDAGLF 0.600 24 142RMAVDLPQDI 0.600 25 257 DEQSDWFYEK 0.486 26 323 RLHGMSSKNI 0.450 27 301ILTGSFPLMS 0.360 28 117 PLWHESDFAV 0.300 29 493 SATTTPNAGK 0.300 30 177YQEFTKNKVK 0.300 Echoed User Peptide Sequence (length = 504 residues)(SEQ ID NO:2)   1 MEELVHDLVS ALEESSEQAR GGFAETGDHS RSISCPLKRQ ARKRRGRKRR 51 SYNVHHPWET GHCLSEGSDS SLEEPSKDYR ENHNNNKKDH SDSDDQMLVA 101KRRPSSNLNN NVRGKRPLWH ESDFAVDNVG NRTLRRRRKV KRMAVDLPQD 151 ISNKRTMTQPPEGCRDQDMD SDRAYQYQEF TKNKVKKRKL KIIRQGPKIQ 201 DEGVVLESEE TNQTNKDKMECEEQKVSDEL MSESDSSSLS STDAGLFTND 251 EGRQGDDEQS DWFYEKESGG ACGITGVVPWWEKEDPTELD KNVPDPVFES 301 ILTGSFPLMS HPSRRGFQAR LSRLHGMSSK NIKKSGGTPTSMVPIPGPVG 351 NKRMVHFSPD SHHHDHWFSP GARTEHDQHQ LLRDNRAERG HKKNCSVRTA401 SRQTSMHLGS LCTGDIKRRR KAAPLPGPTT AGFVGENAQP ILENNIGNRM 451LQNMGWTPGS GLGRDGKGIS EPIQANQRPK GLGLGFPLPK STSATTTPNA 501 GKSA Eachpeptide is a portion of SEQ ID NO: 2; each start position is specified,the length of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine.

TABLE 9A HLA Peptide Motif Search Results User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested 30 HLA molecule type selected A_1101 lengthselected for subsequences to be scored 9 echoing mode selected for inputsequence Y echoing format Numbered lines length of user's input peptidesequence 504 number of subsequence scores calculated 496 number oftop-scoring subsequences reported 30 back in scoring output table

TABLE 9B HLA Peptide Scoring Results - 84P2A9 - A11 9-mers ScoringResults Subsequence Score (Estimate of Half Start Residue Time ofDisassociation of a Molecule Rank Position Listing Containing ThisSubsequence) 1 326 GMSSKNIKK 2.400 2 174 AYQYQEFTK 1.200 3 494 ATTTPNAGK1.000 4 128 NVGNRTLRR 0.800 5 245 GLFTNDEGR 0.480 6 456 WTPGSGLGR 0.4007 258 EQSDWFYEK 0.360 8 283 KEDPTELDK 0.360 9 35 CPLKRQARK 0.300 10 133TLRRRRKVK 0.200 11 176 QYQEFTKNK 0.200 12 157 MTQPPEGCR 0.200 13 40QARKRRGRK 0.200 14 80 RENHNNNKK 0.180 15 410 SLCTGDIKR 0.160 16 210ETNQTNKDK 0.150 17 146 DLPQDISNK 0.120 18 184 KVKKRKLKI 0.120 19 180FTKNKVKKR 0.100 20 409 GSLCTGDIK 0.090 21 381 LLRDNRAER 0.080 22 441ILENNIGNR 0.080 23 482 LGLGFPLPK 0.060 24 459 GSGLGRDGK 0.060 25 275TGVVPWWEK 0.060 26 139 KVKRMAVDL 0.060 27 208 SEETNQTNK 0.060 28 306FPLMSHPSR 0.060 29 178 QEFTKNKVK 0.060 30 179 EFTKNKVKK 0.060 EchoedUser Peptide Sequence (length = 504 residues) (SEQ ID NO:2)   1MEELVHDLVS ALEESSEQAR GGFAETGDHS RSISCPLKRQ ARKRRGRKRR  51 SYNVHHPWETGHCLSEGSDS SLEEPSKDYR ENHNNNKKDH SDSDDQMLVA 101 KRRPSSNLNN NVRGKRPLWHESDFAVDNVG NRTLRRRRKV KRMAVDLPQD 151 ISNKRTMTQP PEGCRDQDMD SDRAYQYQEFTKNKVKKRKL KIIRQGPKIQ 201 DEGVVLESEE TNQTNKDKME CEEQKVSDEL MSESDSSSLSSTDAGLFTND 251 EGRQGDDEQS DWFYEKESGG ACGITGVVPW WEKEDPTELD KNVPDPVFES301 ILTGSFPLMS HPSRRGFQAR LSRLHGMSSK NIKKSGGTPT SMVPIPGPVG 351NKRMVHFSPD SHHHDHWFSP GARTEHDQHQ LLRDNRAERG HKKNCSVRTA 401 SRQTSMHLGSLCTGDIKRRR KAAPLPGPTT AGFVGENAQP ILENNIGNRM 451 LQNMGWTPGS GLGRDGKGISEPIQAMQRPK GLGLGFPLPK STSATTTPNA 501 GKSA Each peptide is a portion ofSEQ ID NO: 2; each start position is specified, the length of peptide is9 amino acids, and the end position for each peptide is the startposition plus eight.

TABLE 10A HLA Peptide Motif Search Results User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested 30 HLA molecule type selected A_1101 lengthselected for subsequences to be scored 10 echoing mode selected forinput sequence Y echoing format Numbered lines length of user's inputpeptide sequence 504 number of subsequence scores calculated 495 numberof top-scoring subsequences reported 30 back in scoring output table

TABLE 10B HLA Peptide Scoring Results - 84P2A9 - A11 10-mers ScoringResults Subsequence Score (Estimate of Half Start Residue Time ofDisassociation of a Molecule Rank Position Listing Containing ThisSubsequence) 1 173 PAYQYQEFTK 3.600 2 481 GLGLGFPLPK 2.400 3 132RTLRRRRKVK 2.250 4 274 ITGVVPWWEK 2.000 5 39 RQARKRRGRK 1.800 6 189KLKIIRQGPK 1.200 7 175 YQYQEFTKNK 0.600 8 180 FTKNKVKKRK 0.500 9 177YQEFTKNKVK 0.300 10 343 VPIPGPVGNK 0.300 11 493 SATTTPNAGK 0.200 12 34SCPLKRQARK 0.200 13 178 QEFTKNKVKK 0.120 14 78 DYRENHNNNK 0.120 15 380QLLRDNRAER 0.120 16 22 GFAETGDHSR 0.120 17 412 CTGDIKRRRK 0.100 18 133TLRRRRKVKR 0.080 19 71 SLEEPSKDYR 0.080 20 325 HGMSSKNIKK 0.080 21 107NLNNNVRGKR 0.080 22 11 ALEESSEQAR 0.080 23 156 TMTQPPEGCR 0.080 24 182KNKVKKRKLK 0.060 25 216 KDKMECEEQK 0.060 26 383 RDNRAERGHK 0.060 27 306FPLMSHPSRR 0.060 28 128 NVGNRTLRRR 0.040 29 111 NVRGKRPLWH 0.040 30 311HPSRRGFQAR 0.040 Echoed User Peptide Sequence (length = 504 residues)(SEQ ID NO:2)   1 MEELVHDLVS ALEESSEQAR GGFAETGDHS RSISCPLKRQ ARKRRGRKRR 51 SYNVHHPWET GHCLSEGSDS SLEEPSKDYR ENHNNNKKDH SDSDDQMLVA 101KRRPSSNLNN NVRGKRPLWH ESDFAVDNVG NRTLRRRRKV KRMAVDLPQD 151 ISNKRTMTQPPEGCRDQDMD SDRAYQYQEF TKNKVKKRKL KIIRQGPKIQ 201 DEGVVLESEE TNQTNKDKMECEEQKVSDEL MSESDSSSLS STDAGLFTND 251 EGRQGDDEQS DWFYEKESGG ACGITGVVPWWEKEDPTELD KNVPDPVFES 301 ILTGSFPLMS HPSRRGFQAR LSRLHGMSSK NIKKSGGTPTSMVPIPGPVG 351 NKRMVHFSPD SHHHDHWFSP GARTEHDQHQ LLRDNRAERG HKKNCSVRTA401 SRQTSMHLGS LCTGDIKRRR KAAPLPGPTT AGFVGENAQP ILENNIGNRM 451LQNMGWTPGS GLGRDGKGIS EPIQANQRPK GLGLGFPLPK STSATTTPNA 501 GKSA Eachpeptide is a portion of SEQ ID NO: 2; each start position is specified,the length of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine.

TABLE 11A HLA Peptide Motif Search Results User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested 30 HLA molecule type selected A24 lengthselected for subsequences to be scored 9 echoing mode selected for inputsequence Y echoing format Numbered lines length of user's input peptidesequence 504 number of subsequence scores calculated 496 number oftop-scoring subsequences reported 30 back in scoring output table

TABLE 11B HLA Peptide Scoring Results - 84P2A9 - A24 9-mers ScoringResults Subsequence Score (Estimate of Half Start Residue Time ofDisassociation of a Molecule Rank Position Listing Containing ThisSubsequence) 1 316 GFQARLSRL 30.000 2 480 KGLGLGFPL 14.400 3 198KIQDEGVVL 14.400 4 373 RTEHDQHQL 12.000 5 182 KNKVKKRKL 8.800 6 139KVKRMAVDL 8.000 7 263 FYEKESGGA 7.500 8 78 DYRENHNNN 7.200 9 300SILTGSFPL 6.000 10 474 QAMQRPKGL 6.000 11 116 RPLWHESDF 6.000 12 110NNVRGKRPL 6.000 13 231 MSESDSSSL 6.000 14 434 VGENAQPIL 6.000 15 64LSEGSDSSL 6.000 16 443 ENNIGNRML 6.000 17 4 LVHDLVSAL 5.760 18 29HSRSISCPL 5.600 19 56 HPWETGHCL 4.800 20 90 HSDSDDQML 4.800 21 478RPKGLGLGF 4.800 22 476 MQRPKGLGL 4.800 23 400 ASRQTSMHL 4.000 24 454MGWTPGSGL 4.000 25 238 SLSSTDAGL 4.000 26 403 QTSMHLGSL 4.000 27 191KIIRQGPKI 3.300 28 349 VGNKRMVHF 3.000 29 15 SSEQARGGF 3.000 30 143MAVDLPQDI 2.592 Echoed User Peptide Sequence (length = 504 residues)(SEQ ID NO:2)   1 MEELVHDLVS ALEESSEQAR GGFAETGDHS RSISCPLKRQ ARKRRGRKRR 51 SYNVHHPWET GHCLSEGSDS SLEEPSKDYR ENHNNNKKDH SDSDDQMLVA 101KRRPSSNLNN NVRGKRPLWH ESDFAVDNVG NRTLRRRRKV KRMAVDLPQD 151 ISNKRTMTQPPEGCRDQDMD SDRAYQYQEF TKNKVKKRKL KIIRQGPKIQ 201 DEGVVLESEE TNQTNKDKMECEEQKVSDEL MSESDSSSLS STDAGLFTND 251 EGRQGDDEQS DWFYEKESGG ACGITGVVPWWEKEDPTELD KNVPDPVFES 301 ILTGSFPLMS HPSRRGFQAR LSRLHGMSSK NIKKSGGTPTSMVPIPGPVG 351 NKRMVHFSPD SHHHDHWFSP GARTEHDQHQ LLRDNRAERG HKKNCSVRTA401 SRQTSMHLGS LCTGDIKRRR KAAPLPGPTT AGFVGENAQP ILENNIGNRM 451LQNMGWTPGS GLGRDGKGIS EPIQAMQRPK GLGLGFPLPK STSATTTPNA 501 GKSA Eachpeptide is a portion of SEQ ID NO: 2; each start position is specified,the length of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight.

TABLE 12A HLA Peptide Motif Search Results User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested 30 HLA molecule type selected A24 lengthselected for subsequences to be scored 10 echoing mode selected forinput sequence Y echoing format Numbered lines length of user's inputpeptide sequence 504 number of subsequence scores calculated 495 numberof top-scoring subsequences reported 30 back in scoring output table

TABLE 12B HLA Peptide Scoring Results - 84P2A9 - A24 10-mers ScoringResults Subsequence Score (Estimate of Half Start Residue Time ofDisassociation of a Molecule Rank Position Listing Containing ThisSubsequence) 1 297 VFESILTGSF 18.000 2 373 RTEHDQHQLL 14.400 3 176QYQEFTKNKV 11.880 4 174 AYQYQEFTKN 9.900 5 432 GFVGENAQPI 9.000 6 51SYNVHHPWET 8.250 7 402 RQTSMHLGSL 8.000 8 315 RGFQARLSRL 8.000 9 263FYEKESGGAC 7.500 10 3 ELVHDLVSAL 7.200 11 280 WWEKEDPTEL 6.600 12 237SSLSSTDAGL 6.000 13 299 ESILTGSFPL 6.000 14 475 AMQRPKGLGL 6.000 15 109NNVRGKRPL 6.000 16 230 LMSESDSSSL 4.800 17 293 VPDPVFESIL 4.800 18 433FVGENAQPIL 4.800 19 63 CLSEGSDSSL 4.800 20 125 AVDNVGNRTL 4.000 21 99VAKRRPSSNL 4.000 22 473 IQAMQRPKGL 4.000 23 453 NMGWTPGSGL 4.000 24 399TASRQTSMHL 4.000 25 292 NVPDPVFESI 3.024 26 142 RMAVDLPQDI 2.880 27 437NAQPILENNI 2.592 28 254 QGDDEQSDWF 2.400 29 14 ESSEQARGGF 2.400 30 323RLHGMSSKNI 2.000 Echoed User Peptide Sequence (length = 504 residues)(SEQ ID NO:2)   1 MEELVHDLVS ALEESSEQAR GGFAETGDHS RSISCPLKRQ ARKRRGRKRR 51 SYNVHHPWET GHCLSEGSDS SLEEPSKDYR ENHNNNKKDH SDSDDQMLVA 101KRRPSSNLNN NVRGKRPLWH ESDFAVDNVG NRTLRRRRKV KRMAVDLPQD 151 ISNKRTMTQPPEGCRDQDMD SDRAYQYQEF TKNKVKKRKL KIIRQGPKIQ 201 DEGVVLESEE TNQTNKDKMECEEQKVSDEL MSESDSSSLS STDAGLFTND 251 EGRQGDDEQS DWFYEKESGG ACGITGVVPWWEKEDPTELD KNVPDPVFES 301 ILTGSFPLMS HPSRRGFQAR LSRLHGMSSK NIKKSGGTPTSMVPIPGPVG 351 NKRMVHFSPD SHHHDHWFSP GARTEHDQHQ LLRDNRAERG HKKNCSVRTA401 SRQTSMHLGS LCTGDIKRRR KAAPLPGPTT AGFVGENAQP ILENNIGNRM 451LQNMGWTPGS GLGRDGKGIS EPIQAMQRPK GLGLGFPLPK STSATTTPNA 501 GKSA Eachpeptide is a portion of SEQ ID NO: 2; each start position is specified,the length of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine.

TABLE 13A HLA Peptide Motif Search Results User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested 30 HLA molecule type selected B7 lengthselected for subsequences to be scored 9 echoing mode selected for inputsequence Y echoing format Numbered lines length of user's input peptidesequence 504 number of subsequence scores calculated 496 number oftop-scoring subsequences reported 30 back in scoring output table

TABLE 13B HLA Peptide Scoring Results - 84P2A9 - B7 9-mers ScoringResults Subsequence Score (Estimate of Half Start Residue Time ofDisassociation of a Molecule Rank Position Listing Containing ThisSubsequence) 1 400 ASRQTSMHL 120.000 2 56 HPWETGHCL 80.000 3 476MQRPKGLGL 40.000 4 29 HSRSISCPL 40.000 5 474 QAMQRPKGL 36.000 6 4LVHDLVSAL 20.000 7 139 KVKRMAVDL 20.000 8 100 AKRRPSSNL 18.000 9 423APLPGPTTA 6.000 10 454 MGWTPGSGL 6.000 11 396 SVRTASRQT 5.000 12 196GPKIQDEGV 4.000 13 182 KNKVKKRKL 4.000 14 110 NNVRGKRPL 4.000 15 198KIQDEGVVL 4.000 16 403 QTSMHLGSL 4.000 17 238 SLSSTDAGL 4.000 18 285DPTELDKNV 4.000 19 300 SILTGSFPL 4.000 20 347 GPVGNKRMV 4.000 21 480KGLGLGFPL 4.000 22 417 KRRRKAAPL 4.000 23 443 ENNIGNRML 4.000 24 313SRRGFQARL 4.000 25 18 QARGGFAET 3.000 26 293 VPDPVFESI 2.400 27 295DPVFESILT 2.000 28 311 HPSRRGFQA 2.000 29 433 FVGENAQPI 2.000 30 486FPLPKSTSA 2.000 Echoed User Peptide Sequence (length = 504 residues)(SEQ ID NO:2)   1 MEELVHDLVS ALEESSEQAR GGFAETGDHS RSISCPLKRQ ARKRRGRKRR 51 SYNVHHPWET GHCLSEGSDS SLEEPSKDYR ENHNNNKKDH SDSDDQMLVA 101KRRPSSNLNN NVRGKRPLWH ESDFAVDNVG NRTLRRRRKV KRMAVDLPQD 151 ISNKRTMTQPPEGCRDQDMD SDRAYQYQEF TKNKVKKRKL KIIRQGPKIQ 201 DEGVVLESEE TNQTNKDKMECEEQKVSDEL MSESDSSSLS STDAGLFTND 251 EGRQGDDEQS DWFYEKESGG ACGITGVVPWWEKEDPTELD KNVPDPVFES 301 ILTGSFPLMS HPSRRGFQAR LSRLHGMSSK NIKKSGGTPTSMVPIPGPVG 351 NKRMVHFSPD SHHHDHWFSP GARTEHDQHQ LLRDNRAERG HKKNCSVRTA401 SRQTSMHLGS LCTGDIKRRR KAAPLPGPTT AGFVGENAQP ILENNIGNRM 451LQNMGWTPGS GLGRDGKGIS EPIQAMQRPK GLGLGFPLPK STSATTTPNA 501 GKSA Eachpeptide is a portion of SEQ ID NO: 2; each start position is specified,the length of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight.

TABLE 14A HLA Peptide Motif Search Results User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested 30 HLA molecule type selected B7 lengthselected for subsequences to be scored 10 echoing mode selected forinput sequence Y echoing format Numbered lines length of user's inputpeptide sequence 504 number of subsequence scores calculated 495 numberof top-scoring subsequences reported 30 back in scoring output table

TABLE 14B HLA Peptide Scoring Results - 84P2A9 - B7 10-mers ScoringResults Subsequence Score (Estimate of Half Start Residue Time ofDisassociation of a Molecule Rank Position Listing Containing ThisSubsequence) 1 318 QARLSRLHGM 30.000 2 293 VPDPVFESIL 24.000 3 345IPGPVGNKRM 20.000 4 433 FVGENAQPIL 20.000 5 125 AVDNVGNRTL 18.000 6 99VAKRRPSSNL 18.000 7 399 TASRQTSMHL 12.000 8 475 AMQRPKGLGL 12.000 9 453NMGWTPGSGL 6.000 10 230 LMSESDSSSL 4.000 11 473 IQAMQRPKGL 4.000 12 312PSRRGFQARL 4.000 13 425 LPGPTTAGFV 4.000 14 103 RPSSNLNNNV 4.000 15 109NNNVRGKRPL 4.000 16 63 CLSEGSDSSL 4.000 17 315 RGFQARLSRL 4.000 18 237SSLSSTDAGL 4.000 19 416 IKRRRKAAPL 4.000 20 196 GPKIQDEGVV 4.000 21 299ESILTGSFPL 4.000 22 402 RQTSMHLGSL 4.000 23 3 ELVHDLVSAL 4.000 24 147LPQDISNKRT 2.000 25 116 RPLWHESDFA 2.000 26 278 VPWWEKEDPT 2.000 27 488LPKSTSATTT 2.000 28 292 NVPDPVFESI 2.000 29 45 RGRKRRSYNV 2.000 30 486FPLPKSTSAT 2.000 Echoed User Peptide Sequence (length = 504 residues)(SEQ ID NO:2)   1 MEELVHDLVS ALEESSEQAR GGFAETGDHS RSISCPLKRQ ARKRRGRKRR 51 SYNVHHPWET GHCLSEGSDS SLEEPSKDYR ENHNNNKKDH SDSDDQMLVA 101KRRPSSNLNN NVRGKRPLWH ESDFAVDNVG NRTLRRRRKV KRMAVDLPQD 151 ISNKRTMTQPPEGCRDQDMD SDRAYQYQEF TKNKVKKRKL KIIRQGPKIQ 201 DEGVVLESEE TNQTNKDKMECEEQKVSDEL MSESDSSSLS STDAGLFTND 251 EGRQGDDEQS DWFYEKESGG ACGITGVVPWWEKEDPTELD KNVPDPVFES 301 ILTGSFPLMS HPSRRGFQAR LSRLHGMSSK NIKKSGGTPTSMVPIPGPVG 351 NKRMVHFSPD SHHHDHWFSP GARTEHDQHQ LLRDNRAERG HKKNCSVRTA401 SRQTSMHLGS LCTGDIKRRR KAAPLPGPTT AGFVGENAQP ILENNIGNRM 451LQNMGWTPGS GLGRDGKGIS EPIQAMQRPK GLGLGFPLPK STSATTTPNA 501 GKSA Eachpeptide is a portion of SEQ ID NO: 2; each start position is specified,the length of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine.

TABLE 15A HLA Peptide Motif Search Results User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested 30 HLA molecule type selected B_3501 lengthselected for subsequences to be scored 9 echoing mode selected for inputsequence Y echoing format Numbered lines length of user's input peptidesequence 504 number of subsequence scores calculated 496 number oftop-scoring subsequences reported 30 back in scoring output table

TABLE 15B HLA Peptide Scoring Results - 84P2A9 - B35 9-mers ScoringResults Subsequence Score (Estimate of Half Start Residue Time ofDisassociation of a Molecule Rank Position Listing Containing ThisSubsequence) 1 478 RPKGLGLGF 120.000 2 56 HPWETGHCL 40.000 3 116RPLWHESDF 40.000 4 425 LPGPTTAGF 20.000 5 334 KSGGTPTSM 20.000 6 400ASRQTSMHL 15.000 7 29 HSRSISCPL 15.000 8 196 GPKIQDEGV 12.000 9 285DPTELDKNV 8.000 10 239 LSSTDAGLF 7.500 11 139 KVKRMAVDL 6.000 12 223EQKVSDELM 6.000 13 488 LPKSTSATT 6.000 14 198 KIQDEGVVL 6.000 15 182KNKVKKRKL 6.000 16 309 MSHPSRRGF 5.000 17 360 DSHHHDHWF 5.000 18 50RSYNVHHPW 5.000 19 103 RPSSNLNNN 4.000 20 468 GISEPIQAM 4.000 21 347GPVGNKRMV 4.000 22 398 RTASRQTSM 4.000 23 295 DPVFESILT 3.000 24 476MQRPKGLGL 3.000 25 74 EPSKDYREN 3.000 26 474 QAMQRPKGL 3.000 27 143MAVDLPQDI 2.400 28 184 KVKKRKLKI 2.400 29 293 VPDPVFESI 2.400 30 90HSDSDDQML 2.250 Echoed User Peptide Sequence (length = 504 residues)(SEQ ID NO:2)   1 MEELVHDLVS ALEESSEQAR GGFAETGDHS RSISCPLKRQ ARKRRGRKRR 51 SYNVHHPWET GHCLSEGSDS SLEEPSKDYR ENHNNNKKDH SDSDDQMLVA 101KRRPSSNLNN NVRGKRPLWH ESDFAVDNVG NRTLRRRRKV KRMAVDLPQD 151 ISNKRTMTQPPEGCRDQDMD SDRAYQYQEF TKNKVKKRKL KIIRQGPKIQ 201 DEGVVLESEE TNQTNKDKMECEEQKVSDEL MSESDSSSLS STDAGLFTND 251 EGRQGDDEQS DWFYEKESGG ACGITGVVPWWEKEDPTELD KNVPDPVFES 301 ILTGSFPLMS HPSRRGFQAR LSRLHGMSSK NIKKSGGTPTSMVPIPGPVG 351 NKRMVHFSPD SHHHDHWFSP GARTEHDQHQ LLRDNRAERG HKKNCSVRTA401 SRQTSMHLGS LCTGDIKRRR KAAPLPGPTT AGFVGENAQP ILENNIGNRM 451LQNMGWTPGS GLGRDGKGIS EPIQAMQRPK GLGLGFPLPK STSATTTPNA 501 GKSA Eachpeptide is a portion of SEQ ID NO: 2; each start position is specified,the length of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight.

TABLE 16A HLA Peptide Motif Search Results User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested 30 HLA molecule type selected B_3501 lengthselected for subsequences to be scored 10 echoing mode selected forinput sequence Y echoing format Numbered lines length of user's inputpeptide sequence 504 number of subsequence scores calculated 495 numberof top-scoring subsequences reported 30 back in scoring output table

TABLE 16B HLA Peptide Scoring Results - 84P2A9 - B35 10-mers ScoringResults Subsequence Score (Estimate of Half Start Residue Time ofDisassociation of a Molecule Rank Position Listing Containing ThisSubsequence) 1 345 IPGPVGNKRM 40.000 2 70 SSLEEPSKDY 20.000 3 196GPKIQDEGVV 18.000 4 318 QARLSRLHGM 18.000 5 14 ESSEQARGGF 10.000 6 99VAKRRPSSNL 9.000 7 103 RPSSNLNNNV 8.000 8 116 RPLWHESDFA 6.000 9 488LPKSTSATTT 6.000 10 293 VPDPVFESIL 6.000 11 299 ESILTGSFPL 5.000 12 237SSLSSTDAGL 5.000 13 467 KGISEPIQAM 4.000 14 56 HPWETGHCLS 4.000 15 147LPQDISNKRT 4.000 16 425 LPGPTTAGFV 4.000 17 358 SPDSHHHDHW 3.000 18 230LMSESDSSSL 3.000 19 253 RQGDDEQSDW 3.000 20 399 TASRQTSMHL 3.000 21 184KVKKRKLKII 2.400 22 445 NIGNRMLQNM 2.000 23 402 RQTSMHLGSL 2.000 24 300SILTGSFPLM 2.000 25 334 KSGGTPTSMV 2.000 26 433 FVGENAQPIL 2.000 27 210ETNQTNKDKM 2.000 28 63 CLSEGSDSSL 2.000 29 486 FPLPKSTSAT 2.000 30 315RGFQARLSRL 2.000 Echoed User Peptide Sequence (length = 504 residues)(SEQ ID NO:2)   1 MEELVHDLVS ALEESSEQAR GGFAETGDHS RSISCPLKRQ ARKRRGRKRR 51 SYNVHHPWET GHCLSEGSDS SLEEPSKDYR ENHNNNKKDH SDSDDQMLVA 101KRRPSSNLNN NVRGKRPLWH ESDFAVDNVG NRTLRRRRKV KRMAVDLPQD 151 ISNKRTMTQPPEGCRDQDMD SDRAYQYQEF TKNKVKKRKL KIIRQGPKIQ 201 DEGVVLESEE TNQTNKDKMECEEQKVSDEL MSESDSSSLS STDAGLFTND 251 EGRQGDDEQS DWFYEKESGG ACGITGVVPWWEKEDPTELD KNVPDPVFES 301 ILTGSFPLMS HPSRRGFQAR LSRLHGMSSK NIKKSGGTPTSMVPIPGPVG 351 NKRMVHFSPD SHHHDHWFSP GARTEHDQHQ LLRDNRAERG HKKNCSVRTA401 SRQTSMHLGS LCTGDIKRRR KAAPLPGPTT AGFVGENAQP ILENNIGNRM 451LQNMGWTPGS GLGRDGKGIS EPIQAMQRPK GLGLGFPLPK STSATTTPNA 501 GKSA Eachpeptide is a portion of SEQ ID NO: 2; each start position is specified,the length of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine.

1. An isolated polynucleotide that encodes the polypeptide sequence ofSEQ ID NO:2.
 2. An isolated polynucleotide comprising the sequence setforth in SEQ ID NO:1 from nucleotide residue number 720 throughnucleotide residue number
 1392. 3. A recombinant expression vector thatcontains the polynucleotide of claim
 1. 4. The recombinant expressionvector of claim 3, wherein the polynucleotide encodes the polypeptidesequence of SEQ ID NO:2.
 5. A recombinant expression vector thatcontains the polynucleotide of claim
 2. 6. The recombinant expressionvector of claim 3, wherein the vector is a viral vector.
 7. Therecombinant expression vector of claim 6, wherein the viral vector isderived from a virus selected from the group consisting of vaccinia,fowlpox, canarypox, adenovirus, influenza, poliovirus, adeno-associatedvirus, lentivirus, and sindbus virus.
 8. The recombinant expressionvector of claim 6, wherein the viral vector is derived from canarypox.9. The recombinant expression vector of claim 6, wherein the viralvector is derived from adenovirus.
 10. The isolated polynucleotide ofclaim 1, wherein the polynucleotide is encoded by a plasmid designatedp129.1-US-P1 and deposited with American Type Culture Collection asAccession No. PTA-1151.